Micriobiology Flashcards by Kaori Chan

BSC CLASSES:
In at front through HEPA to the outside or into the room through HEPA With possible contamination

BSC I

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BSC CLASSES:
NO AIR RECIRCULATION; total exhaust to the outside
With possible contamination

I B2

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BSC CLASSES:
70% recirculated, 30% exhausted

BSC II A1

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BSC CLASSES:
same as II A1 but has 100 FLM (linear feet per min)
Vertical Laminar Flow: air flows in sheets.
BSC IIA or IIB are recommended in school/hospitals

BSC II A2

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BSC CLASSES:
70% EXHAUSTED; 30% RECIRCULATED
Exhaust air outside the building; for radioisotopes, carcinogens, & toxic chemicals
Vertical Laminar Flow: air flows in sheets.
BSC IIA or IIB are recommended in school/hospitals

II B1

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BSC CLASSES:
SUPPLY and EXHAUST air thru HEPA. Close cabinet for BSL-4 Research facilities

III

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Certification of Biosafety Cabinet should be done after:

Installation ;
If Repositioned more than 18 inches ; or
Annually, or as indicated by risk assessment.

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PHYSICAL METHODS OF STERILIZATION: HEAT
MOIST:
Autoclave “Steam under pressure”
Tyndallization / Fractional sterilization
Inspissation

DRY HEAT:
Oven

121°C 15 psi for15-30 mins or 132°C 15 psi 30-60 min (for sterilization)

100°C 15 psi for 30 mins for 3 consecutive days (1st: vegetative, 2nd: spores, 3rd: remaining cells)

70-80°C for 2 hours 3 consecutive days (for heat sensitive, Lowenstein, Jensen)

160-170°C for 1.5-3 hours For drying; Glassware, cotton swab, oils,
petrolatum, powder

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PHYSICAL METHODS OF STERILIZATION:
870-980degC
For research animals and medical/laboratory waste; safest
method to ensure no infective materials remain in samples

INCINERATION

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PHYSICAL METHODS OF STERILIZATION: FILTRATION
Pulling liquids through a membrane (cellulose acetate or cellulose
nitrate) with a vacuum; For heat-sensitive materials like
antibiotic solutions, toxic chemicals, radioisotopes, vaccines, carbohydrates. Usual pore size: ___ Critical sterilizing for parenteral
solutions: 0.22 μm Filtering small viruses: 0.1 μm

has a 0.3um filtration; Used in isolation rooms, operating
rooms, biological safety cabinets

Membrane filtration; 0.45-0.80 μm

HEPA filter

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PHYSICAL METHODS OF STERILIZATION:
Composed of short wavelength, high energy gamma rays
Used in sterilizing disposables such as plastic syringe,
vacutainer blood tubes, catheters, gloves

IONIZING/GAMMA
RADIATION

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CHEMICAL METHODS OF STERILIZATION:
For heat-sensitive materials
Disadvantage: lengthy cycle times and health hazard

ETHYLENE OXIDE (ETO)

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CHEMICAL METHODS OF STERILIZ ATION:
Oxidizing agent; Used in sterilizing HEPA filter in BSCs, metals, and
medical instruments like scissors. No toxic by-products

VAPOR-PHASE HYDROGEN PEROXIDE

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CHEMICAL METHODS OF STERILIZATION:
H2O2 generates plasma by exciting the gas in enclosed chamber under deep vacuum with the use of radiofrequency or microwave energy

HYDROGEN PEROXIDE GAS PLASMA

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PHYSICAL METHODS OF DISINFECTION: PASTEURIZATION
___: method 63degC, 30 min
___: method 72degC 15 sec Bailey’s: 70degC 30 min

kills food pathogens without
damaging the nutritional value or flavor. Properly pasteurized milk should have a ___ ALP/Phosphatase test.

Batch
Flash

negative

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ANTISEPTICS:
Tincture of iodine: ___ + ___
70% ethyl alcohol followed by iodophor used in drawing blood culture and surgery.

___: Iodine + Neutral polymer (Povidone-Iodine, Poloxamer-
Iodine)
Contact time for must be >30seconds

Iodine must be removed completely from the skin to avoid irritation. Must be diluted properly. Not sporicidal

HALOGEN- Iodine
Iodine + Alcohol

Iodophor

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ANTISEPTICS: PHENOLICS
Has high
antimicrobial activity better than iodine) and has low toxicity.
More effective on gram(+) than in gram(-)
Less effective in Fungi and Mycobacteria; Naked viruses are not inactivated.

Chlorhexidine gluconate (CHG)

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Biological Indicator (Bioindicators) of Sterilization: Autoclave

Geobacillus (Bacillus) stearothermophilus

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Biological Indicator (Bioindicators) of Sterilization: Dry heat/Oven

Bacillus subtilis
Bacillus atrophaeus

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Biological Indicator (Bioindicators) of Sterilization: Radiation

Bacillus pumilus

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Biological Indicator (Bioindicators) of Sterilization: Gas sterilant or
Ethylene oxide

Bacillus subtilis
Bacillus atrophaeus

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Most commonly used autoclave:

Gravity Displacement Autoclave

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most resitant to most sensitive order of microbes

Prions > Endospore > Mycobacteria > Naked/Nonlipid viruses > Fungi > Bacteria > Enveloped/Lipid viruses

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Methods of Killing of Prions:
Autoclave: ___ in prevacuum sterilizer
__ for 1 hour in Gravity displacement sterilizer
1N NaOH: __ for 1 hour then autoclave

134degC for 18 minutes
132degC

Immersion

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___: Single cellular except Cyanobacteria 2 Domains: 1. Bacteria (Environmental cyanobacteria/blue-green algae and Medically relevant bacteria); and 2. Archaea (Extreme habitats) high salt conc, jet fuel, high temperature Has circular chromosome; Cell wall has peptidoglycan

Prokaryotes (Prenucleus)

__: Fungi and Parasites Single or Multicellular organisms Has linear chromosome Cell wall has NO peptidoglycan.

Eukaryotes (True nucleus)

__: Viruses - Host-dependent infectious agents Prions – Abnormal infectious proteins TAXA: order?

Infectious Particles (do kings play chess on fine green silk) Domain – Kingdom – Phylum – Class – Order – Family – Genus – Species

Classification, nomenclature, and identification of organisms Deals with variations in gene expression not caused by nucleic acid sequence similarities or differences It is a method of organizing microorganisms into groups or taxa based on similar morphologic, physiologic, and genetic traits.

taxonomy epigenetics classification

Is the process consisting of a series of methods designed to provide the microbiologist with relevant and useful clinical information about a microorganism Naming of microorganisms according to rules of International Code of Nomenclature of Prokaryotes (ICNP). Is considered the same species with the same genetic make-up but displays differential physiologic characteristics.

identification Nomenclature Biotype / biovar

A strain of microorganism that can be differentiated based on antigen types using specific antibodies. A specific version of an organism; a subtype of a specific organism with some minor differences Characterization of etiologic agents using molecular techniques important during an outbreak or epidemic.

serotype/serovar Strain Strain typing

Macroscopic and Microscopic morphology, Staining characteristics, Environmental & Nutritional requirements, Resistance profiles, Antigenic and Subcellular properties, Chemotaxonomic properties (teichoic acid, fatty acid, protein, cell wall composition) DNA base composition ratio (guanine, cytosine, adenine, thymine), Nucleic acid base sequence (hybridization tests), Average nucleotide identity (ANI), Genome-to-genome distance

phenotypic criteria genotypic criteria

the ability of the microorganism to produce disease in a host. the degree of damage caused by the microorganism to the host; degree of pathogenicity. growth of microorganisms that result in damage to host. Ingestion of preformed bacterial toxins present in food.

Pathogenicity Virulence Infection Intoxication

Infection is transmitted by direct contact between reservoir and host Infection is transmitted to host via intervening agents (Vectors or Vehicles) Results when infection produces notable changes in human physiology associated with damage or loss of function to one or more of the body’s organ system.

Direct transmission Indirect transmission Disease

A result of regular interaction with animals for food production A larger than normal number of diseased or infected individuals that occurs over a relatively short period Any type of epidemiologic investigation that involves data collection for characterizing circumstances surrounding the incidence or prevalence of a particular disease or infection.

Livestock-associated infection Outbreak Surveillance

Pioneers in the History of Microbiology: Father of microbiology; 1st microbiologist, 1st to describe bacteria phenol as antimicrobial Father of Epidemiology proposed heat in killing bacteria (aseptic technique) and pasteurization

Antonie van Leeuwenhoek Joseph Lister John Snow Louis Pasteur

Pioneers in the History of Microbiology: Father of Taxonomy Germ theory; Anthrax & Tuberculosis Father of Handwashing/Hospital hygiene Endospore survival on boiling

Carl von Linne Robert Koch Ignaz Semmelweis FerdinandCohn

Pioneers in the History of Microbiology: Father of virology (discovery of virus); use of enrichment and selective media Theory of chemolithotrophs; use of enrichment and selective media Discovered HIV that causes AIDS Developed >40 vaccines: against Hepatitis A and B, Measles, Mumps, Rubella,S. meningitidis, S. pneumoniae

Martinus Beijerinck Sergei Winogradsky Luc Montagnier Maurice Hilleman

Pioneers in the History of Microbiology: Developed Gram stain technique Inventor of PCR Petri developed petri dish use of agar as culture media instead of gelatin

Hans Christian Gram Karry Mullis Julius Richard Fanny Hesse

BACTERIAL STRUCTURE: Cell wall - Morphology determinant: cocci, coccobacilli, bacilli, curved, fusiform, spiral shapes - Primary Basis of __ - Has PEPTIDOGLYCAN/______________ LAYER - Composed of N-acetyl-D-glucosamine (NAG) & N-acetyl-D-muramic acid (NAM)

bacterial classification MUREIN

GRAM POSITIVE Outer membrane and Periplasmic space: Peptidoglycan Negative charge source Flagella Physical destruction B-lactam antibiotic Basic dyes

Absent T H I C K Teichoic Acid 2 Basal Rings Resistant Sensitive Sensitive

GRAM NEGATIVE Outer membrane and Periplasmic space Peptidoglycan Negative charge source Flagella Physical destruction B-lactam antibiotic Basic dyes

Present Thin Lipopolysaccharide (LPS) 4 Basal Rings Sensitive Resistant Resistant

Most important DIFFERENTIAL staining test in Microbiology Stains the microorganism’s cell wall. Most critical step – ___ Iodine should be BASIC. Because dyes are ____________ /_____________.

GRAM STAIN decolorizer cationic/basic

Reagents of GRAM STAINING (mnemonic) 1° stain Mordant Decolorizer 2°Counterstain

(VIAS) crystal violet Gram's iodine 95% alcohol/acetone safranin

QC ORGANISMS FOR GRAM STAIN: Gram Positive; Dark purple to Deep blue: ____ Gram Negative; Pink to Deep Magenta: ___

Staphylococcus aureus Escherichia coli

Resists Decolorization: __ (4) (may appear as Gram-positive) Appears as Gram Neutral/ Gram: __ (Poorly stained) Poorly stained with Gram stain: (3) Poorly stained with Safranin: (4) NOT STAINED ___ (Chlamydia, Rickettsia, Ehrlichia, Coxiella), __ (Mycoplasma/Ureaplasma), for Dark-field microscopy (__)

Acinetobacter, Kingella, Psychrobacter, Methylobacterium Mycobacteria (Poorly stained) Campylobacter, Brucella, Tropheryma whipplei Legionella, Gram negative anaerobes, Francisella Intracellular; No cell wall; Spirochetes

__: Screening of quality is thru GRAM STAIN: Bartlett’s classification, <10 epithelial cells, >25 pus cells under LPO ______________: (2x3 cm smear; 300 OIF is examined)

SPUTUM DSSM

Demonstrates the presence of Mycolic Acid or Hydroxymethoxy Acid in bacterial cell wall. Confirms suspected TB cases if patient is symptomatic or CXR is suggestive

ACID FAST STAIN

Hot method: __ Cold method: __ BEST Routine AFS method: __ BEST Tissue AFS method: __ MOST Sensitive AFS method: __ (read 30 LPF)

Ziehl-Neelsen Kinyoun Ziehl-Neelsen Kinyoun Auramine-Rhodamine

Ziehl-Neelsen (MNEMONIC) 1° stain: Mordant: Decolorizer: 2°/Counterstain: Acid fast: Non-Acid fast::

(CHAM) Carbolfuchsin Heat 3% Acid Alcohol Methylene blue Red/Pink Blue

Kinyoun 1° stain: Mordant: Decolorizer 3%: 2°/Counterstain: Acid fast: Non-Acid fast:

Carbolfuchsin Phenol/Tergitol 3% Acid Alcohol Methylene blue or Malachite green Red/Pink Green

Auramine-Rhodamine: 1° stain: Mordant: Decolorizer 3%: 2°/Counterstain: Acid fast: Non-Acid fast:

Auramine-Rhodamine no mordant 0.5% acid none Yellow fluorescence No fluorescence

Auramine-Rhodamine quenching agent ________; 3% Acid alcohol is composed of 3% HCl and 95% __. AFB smear sensitivity: __ Sensitivity of Acid fast stain is 5,000 – 10,000 AFB/mL Newly required number of sputum sample for TB detection: ___ Spot-Spot / Frontloading: 2 samples: __ apart 1. __: 2 samples; 1 random & 1 early morning sputum ~1/2 or 2/2 positive AFB sample → A confirmed case of tuberculosis.

0.5% potassium permanganate (0.5% KMNO4) Ethanol 20-80% 2 sample one-hour Spot-AM / Spot-Early morning

CDC AFB Grading: No AFB seen 0 AFB/__ fields Indeterminate 1-2 AFB/__fields 1+ 1-9 AFB/__ fields 2+ 1-9 AFB/__ fields (at least 50 fields) 3+ 1-9 AFB/field 4+ __ AFB/field

300 300 100 10 >9

_______________ (Fading) – permanent loss of fluorescence due to chemical damage of fluorochrome _______________ – a result of transfer of light energy to nearby molecules in the sample such as free radicals, salts of heavy metals, or halogens.

photobleaching quenching

___ staining (urine) –M. smegmatis (Blue) vs M. tuberculosis (Red) ___ staining (tissue) – M. leprae (Red) vs M. tuberculosis (Blue) __ stain (tissue) – for M leprae

Pappenheim’s Baumgarten’s Fite Faraco

__ for PARTIAL ACID-FAST microorganisms like Nocardia, Coccidians Modified because they use __ such as 1% H2SO4 Alcohol. 2a. __ > Outer membrane - found only in Gram __ bacteria - Initial barrier of bacteria to the environment - Composed of Lipopolysaccharide that gives the bacterial surface its ___ charge. > Inner membrane - A permeability barrier - For cell wall synthesis - site of __ synthesis as a counterpart of mitochondria 3a. __ - found only in Gram- negative; has gel-like substance that capture nutrients and detoxifies environmental solutes like antibiotics.

MODIFIED ACID-FAST STAIN weak decolorizer Cytoplasmic membrane negative negative energy/ATP Periplasm/Periplasmic space

2. BACTERIAL DNA A. dsDNA B. __ – EXTRAchromosome that carries antibiotic resistance genes. 3. __ - __Bacteria; __ Fungi; for synthesis of proteins 4. CAPSULE - consists of polysaccharide/carbohydrates except for __ (_______/amino acid,protein) - contributes to bacterial colonization and __ in agar plates - attachment and anti-phagocytic __ = N. meningitidis, S. pneumoniae, K. pneumoniae, H. influenzae, Cryptococcus neoformans Lab dx:___ reaction (Capsular swelling) and K or Vi Ag typing

PLASMID RIBOSOME; 70s; 80s B. anthracis; Poly-D-glutamic acid slime layer/mucoid layer ENCAPSULATED Neufeld Quellung

___ – food reserve, lessens osmotic pressure C. diphtheria: Y. pestis: M. tuberculosis: Nocardia:

METACHROMATIC GRANULES Babes-Ernst Bipolar bodies Much granules Sulfur granules

6. – for attachment (common/somatic pili) (N. gonorrheae, B. pertussis) and for gene transfer (sex pili, F factor) Mechanism of Gene Transfer 1. Transformation: 2. __ – Transposon (jumping genes), plasmid, sex pilus 3. __ – Bacteriophage

PILI OR FIMBRIAE Conjugation Transduction

7. __ – made of Calcium dipicolinate. Resistance to heat, chemicals, nutrient deprivation, drying. Bacillus anthracis (__), Clostridium tetani (__), C. botulinum (__)

ENDOSPORE center; terminal; subterminal

8. __ – for locomotion A-trichous: __ (Non-motile microorganism) __-trichous: single flagellum located at one end of the cell __-trichous: single flagellum located at both ends of the cell __-trichous: group of flagella located at one end of the cell __-trichous: multiple flagella located around the surface of the cell → __ – composed of Axial filament; for spirochete, cork-screw

FLAGELLA no flagella MONO AMPHI LOPHO PERI Endoflagella

type of motility? Kingella, B. henselae Listeria Campylobacter Vibrio Treponema Capnocytophaga Motility: - Best observed at ______ TESTS FOR MOTILITY: ___ (Motility medium) – spreading (+) ___ – motility observed

Twitching Tumbling Darting Rapid Shooting star Cork screw Gliding room temperature SIM Hanging drop test

9. __ – has thousands of enzymes and the site of protein synthesis 10. __ – has the bacterial chromosome which is circular and complexed with RNA (Eukaryotic chromosome is linear and complexed with histones)

CYTOSOL NUCLEOID

___ – are genomic regions in which virulence factors are encoded in. ___ – consists of monomicrobic or polymicrobic group of bacteria housed in a complex biochemical matrix. Adheres to implants and prosthetic devices; contributes to ___. ___ – Starved, Nutrient-deprived, slow-growing state of cells in a biofilm which displays reduced susceptibility to antimicrobial agents. Mechanism of signaling: Gram negative: __ (AHL) and Gram positive: __

Pathogenicity islands (PAIs) Biofilm; antibiotic resistance Persister cells Acyl homoserine lactone; Oligopeptides

__: S. aureus, CONS, Streptococcus, Listeria monocytogenes, P. aeruginosa, Acinetobacter, Aeromonas, Aggregatibacter spp., Salmonella spp., Enterobacter, Enterococcus, Escherichia coli, Klebsiella, Proteus, Serratia, Citrobacter koseri, and Candida albicans. __ – are biochemically active substances that have particular effects on host cells

Biofilm producers Toxins

___; a protein; 100degC heat stability: Destroyed; Formaldehyde: Detoxified Immunologic, convertible to toxoid vaccine is Specific, Localized infection; Kills host cells, interfere in cell signals and protein synthesis

EXOTOXIN: Gram-Positive

Protein-lipid-polysaccharide; 100degC: Intact, released from cell lysis Not Detoxified by formaldehyde; Non-immunologic, not neutralized by antitoxin; Broad; Systemic infection; Either disrupt or form clots (DIC), Fever, Activation of complement/immune system, Hypotension, Shock, Death

ENDOTOXIN: Gram-Negative

___: Immunodiffusion, PCR, Tissue culture, EIA, RIA, Limulus amebocyte lysate ➢ Horseshoe crab’s (Limulus polyphemus) blood cell called __ is separated from plasma and lysing them makes a “lysate”, when mixed with __, it will form clots/precipitates and cause turbidity. ➢ Latex agglutination – for detection of toxins of __, E. coli O157, and __

Toxin testing amebocyte endotoxin Clostridium difficile; Rotavirus

SPECIAL STAINS: ___ - Dyar, Victoria Blue, Calcofluor white (for Fungal chitin) ___ - Levaditi Silver Impregnation, Fontana tribondeau ___ - Toluidine blue

Cell wall Spirochetes Helicobacter pylori

SPECIAL STAINS: ___ - Ziehl-Neelsen, Kinyoun, Auramine-Rhodamine, Pappenheim, Baumgarten, Wadefite, Gabett ___ - Neisser’s, Albert’s, Ljubinsky, LAMB (Loeffler’s Alkaline Methylene Blue), Burke’s ___ - Wayson’s

Acid Fast Metachromatic granules Bipolar bodies

SPECIAL STAINS: ___ - Dorner’s, Wirtz-Conklin, Schaeffer-Fulton ___ - Gray’s, Leifson, Caesares Gil, Fisher-Conn ___ - Anthony’s, Nigrosin, India Ink ___ - McFadyean

Endospore Flagella Capsule/Negative stains Bacillus anthracis

SPECIAL STAINS: ___ - Acridine orange, Feulgen ___ - Gimenez, Macchiavello ___ - Dienes stain

Nucleic acid/DNA Rickettsia Mycoplasma

Schaeffer-Fulton stain, mordant, decolorizer and counterstain

malachite; heat; tap water; safranin

Bacteria’s dry weight is made out of 50% __ and 14% __. __ – uses Carbon dioxide as sole source of carbon A. __ – Use light as source of energy B. __ – uses inorganic compounds as source of energy ___ – require complex substances for growth; requires organic substances for energy; (clinically significant bacteria that causes human infection)

Carbon; Nitrogen. Autotrophs Phototrophs Chemolithotrophs Heterotrophs

TEMP: ___ (0-20degC) ___ contamination (Food-Listeria) & Blood bank- __, P. fluorescens, S. liquifaciens

Psychrophiles/Cryophiles Refrigerator Y. enterocolitica

TEMP: __ (20-40 degC) Room temp contamination Blood Bank - ___, B. cereus Temperature enrichment method A. __ enrichment (4oC) B. __ enrichment (42oC)

Mesophiles S. epidermidis Cold Hot

TEMP: ___ (40-60 degC) Bacillus stearothermophilus Room temperature (20-25oC) – BEST for ___ production; Use Sterile White swab to pick up colony. Incubator temperature – ________; Fungal cultures - (__)

Thermophiles Microbial Pigment 35-37degC; 30degC

pH: Lactobacillus acidophilus Also known as: 1. Doderlein bacilli (Vagina) 2. Boas Oppler bacilli (Gastrointestinal) Vibrio spp.

Acidophilic <5.5 pH Alkalinophilic >8.0 pH

Salt concentration: __ – Salt-Loving bacteria (Requires NaCl for growth) S. aureus – __ NaCl (MSA) Enterococcus – __ NaCl broth Vibrio – 0, 3, 5, 8, 10% NaCl broth (All Vibrio are halophilic except:____________________)

HALOPHILES 7.5%; 6.5% V. cholerae and V. mimicus

21% O2; 0.03% CO2 Usual Incubator, AMBIENT air/ ROOM air

AEROBE FUNGI, MTB, Brucella, Bordetella

ANAEROBE: 0% O2; 5-10% H2; 5-10% CO2; 80%-90% N2 Gas Pak Jar, AnaeroPack Palladium, Brewer jar, McIntosh & Fildes

Obligate anaerobe Clostridium Bacteroides

ANAEROBE: Do not require O2 but grows better with O2 Usual Incubator, AMBIENT air/ ROOM air

Facultative anaerobe MOST pathogens, Enterobacteria

ANAEROBE: Grows at the same rate with or without O2 Usual Incubator, AMBIENT air/ ROOM air

Aerotolerant anaerobe Cutibacterium (Propionibacterium) acnes

5-10% O2 (decreased); 8-10% CO2(increased); 85% N2 Microaerophilic incubator or Jars

MICROAEROPHILE Campylobacter. Helicobacter, Treponema, Borrelia *(sa camp konti oxygen sa loob, tas may ulcer, gumawa ng trap for boars)*

5-10% CO2 (increased) Candle Jar, Capnophilic incubator

CAPNOPHILE Haemophilus, HACEK, Neisseria, S. pneumoniae

SPECIAL GROWTH SUPPLEMENTS: ___: X and V factors, IsoVitale X or Vitox ___: Cysteine and Iron

Haemophilus Legionella

SPECIAL GROWTH SUPPLEMENTS: __: Cysteine and Cystine __: Cysteine, Methionine, Nicotinic acid __: Cysteine and Vitamin B6 (Pyridoxal)

Francisella Bordetella pertussis Granulicatella, Abiotrophia

SPECIAL GROWTH SUPPLEMENTS: __: Hemin and Vitamin K __: Vitamin B1 (thiamine)

Anaerobes (Bacteroides) B. anthracis and Trichophyton fungus

BACTERIAL GROWTH CURVE Generation time: can be every __ (E. coli) to every __ (M. tuberculosis) Phase sensitive to antimicrobials? Phase that has decrease in nutrients, increase in toxins; where sporulation occurs?

30 minutes; 24 hours Exponential/Log phase Stationary/Plateau phase

CULTURE MEDIA. In general: pH: __, distilled/deionized water; Dissolved till clear and no particles seen. Heat-labile or delicate media are sterilized through __ or through __ (pore size 0.2-0.45um) Types of Bacterial Culture: __ (3) NOTES: __ - most common solidifying agent After the media has cooled from sterilization, just before dispensing, add supplements (blood, vitamins, nutrients, or antibiotics) Dispense ___ of cooled (55-60oC) media on each 100 mm petri dish (commonly used).

7.2-7.4 116-118degC; membrane filtration Pure culture, Mixed culture, Stock culture Agarose 20-25 mL

Antimicrobials used in Culture media for Neisseria: What inhibits Gram positive? negative? Fungi?

Vancomycin, Lincomycin, Colistin (VLC is nice) Nystatin, Amphotericin B, Anisomycin

GRAM NEG MEDIUMS: Inhibitory: Eosin Y, Methylene Blue Indicator: Eosin Y, Methylene Blue NLF: Colorless or Translucent ~ E. coli appears as ___ E. aerogenes as fish-eye colony

EMB (Levine agar); green metallic sheen

GRAM NEG MEDIUMS: Inhibitory: Crystal violet, Bile salt Indicator: Neutral red NLF: Colorless ~__ – detection of E.coli O157:H7

MAC Mac Sorbitol

GRAM NEG MEDIUMS: Inhibitory: Bile salt Carbohydrate: Lactose, Xylose, Dextrose Indicator: Phenol red LF: Yellow NLF: Red/Colorless ~ Selective and differential for Salmonella and Shigella

XLD: Xylose Lysine Deoxycholate

GRAM NEG MEDIUMS: Inhibitory: Bile salt, Bromothymol blue, Acid fuchsin Carbohydrate: Lactose, Salicin, Sucrose Indicator: Bromothymol blue LF: Yellow NLF: Blue Green/Colorless ~Differentiates Salmonella and shigella from other enteric gram- negative bacteria

HEA: Hektoen Enteric Agar

GRAM NEG MEDIUMS: Inhibitory: Bile salt Carbohydrate: Lactose Indicator: Neutral red LF: Red/Pink NLF: Colorless ~ For Salmonella and Shigella

DCA: deoxycholate citrate agar

GRAM NEG MEDIUMS: Inhibitory: Brilliant green, bile salt Carbohydrate: Lactose Indicator: Neutral red LF: Red/Pink NLF: Colorless ~ For Salmonella and Shigella

SSA: Salmonella Shigella (SS) Agar

GRAM NEG MEDIUMS: Inhibitory: Brilliant green Carbohydrate: Lactose Indicator: Bismuth sulfite NLF: Black (Salmonella ~ For Salmonella

BSA: Bismuth sulfite agar

GRAM NEG MEDIUMS: Inhibitory: Bile salt Carbohydrate: Sucrose Indicator: Bromothymol blue LF: Yellow (sucrose) NLF: Green (sucrose) ~ For Vibrio

TCBS: Thiosulfate–citrate–bile salts–sucrose agar

CULTURE MEDIA: __: Fastidious and Non-fastidious microorganisms __: For anaerobes, aerobes, microaerophilic and fastidious organisms __: backup/enrichment broth for subculturing bacteria from primary agar plate __: Fastidious and Non-fastidious microorganisms (for sterile samples) __: Non-fastidious microorganisms __: Fastidious organisms (Haemophilus, Neisseria, Brucella)

Nutrient agar Thioglycollate broth Trypticase soy broth Brain-Heart Infusion Agar or Broth Blood Agar Chocolate agar

CULTURE MEDIA: __: Each kind is intended for a specific bacteria like E. coli O157, MRSA, Candida albicans __: Each kind is intended for “bacteria-specific” drug-resistant strains; Carbapenem-resistant, ESBL-positive, VRE, MRSA __: Gram positive cocci and bacilli (aerobic/anaerobic) __: Gram positive cocci __: Staphylococcus aureus Mueller Hinton agar with 2% NaCl or with 4% NaCl and 6 ug Oxacillin: for culturing ___ LIM, Todd-Hewitt broth, carrot medium, Granada medium; Hippurate broth (Identification purpose): for ___

Chromogenic media Brilliance agar Phenylethyl alcohol (PEA) agar Columbia colistin-nalidixic acid (CNA) agar Mannitol salt agar MRSA S. agalactiae

__: Group D streptococci, Enterobacter, Klebsiella, Serratia, Listeria __: Vancomycin-resistant Enterococci __: Enterococci Polymyxin-Acriflavine-Lithium Chloride-Ceftazidime Esculin- Mannitol (PAL-CAM broth): __ Cystine-tellurite blood agar, Loeffler medium, Potassium tellurite agar, Tinsdale agar (TIN agar): __

Bile Esculin Agar (BEA) Bile esculin azide agar with Vancomycin Enterococcosel agar Listeria monocytogenes Corynebacterium

PLET agar (Polymyxin B-lysozyme-EDTA-Thallous acetate): __ MEYP/MYP; PEMBA; BCM: __ 0.4% Dilute gelatin medium: __ Petragnani medium, Lowenstein-Jensen medium, Middlebrook 7H9 (broth): __ Selenite broth, Bismuth sulfite agar, Brilliant green agar: __ Phenylalanine Deaminase agar (PAD): __

Bacillus anthracis Bacillus cereus Nocardia and Streptomyces differentiation Mycobacterium tuberculosis Salmonella (the 2 agars are very selective for Salmonella) Proteus-Morganella-Providencia (PMP group)

__: Yersinia enterocolitica, Aeromonas spp. Cycloserine Cefoxitin Fructose Agar (CCFA): __ Egg yolk agar (EYA) AKA McClung Toabe agar: __

Cefsulodin-Irgasan-Novobiocin (CIN) agar Clostridium difficile Clostridium perfringens

Alkaline Peptone water (APW) and Thiosulfate citrate-bile salts (TCBS) agar: __ __: Pseudomonas aeruginosa Bordet-Gengou Agar, Regan-Lowe: __ __: agar with antibiotics Legionella Wadsworth Brucella Blood agar:__ Blood agar with Kanamycin and Vancomycin (KV blood agar): __

Vibrio cholerae and Vibrio parahaemolyticus Cetrimide agar Bordetella Buffered Charcoal Yeast Extract (BCYE) Moderately-fastidious anaerobes Bacteroides

TRANSPORT MEDIA: Enteric pathogens (Stool samples): __ Neisseria gonorrhoeae and Neisseria meningitidis: __ Viruses: __ Amies and Stuart’s transport media: __ __: Chlamydia, Mycoplasma/Ureaplasma, and Viruses

Cary-Blair JEMBEC media VTM (viral transport medium Aerobic and anaerobic bacteria Universal transport media (UTM)

___ is done by conducting phenotypic and genotypic laboratory tests to gather information and identify the microorganism responsible for the disease. Before starting a work up, a PURE CULTURE must be used to yield a proper identification and then later on, susceptibility testing of the microorganism.

Work up/Identification Scheme

A. __: Use of slides in smear preparation, special staining, and manual biochemical testing. B. __: Use of Analytical Profile Index (API) – for identification of fermentative gram negative bacteria. Has 20 cupules. __ profile number is recorded which is equivalent to a specific bacteria in the database. C. ___: Colorimetry, Fluorometry, or Turbidimetry are used to detect bacterial growth.

Manual/Conventional Method Manual Multitest System; 7-digit code Automated Methods

Automated Methods: MicroScan and TREK Diagnostic System: uses __ __: Uses 64 well cards for identification, sensitivity testing, and detection of multi-drug resistant isolates within 16 to 24 hours including anaerobic organisms. AST results are interpreted using ________________ (AES) within 6-8hours. __: Through colorimetric and fluorometric system, a 136 well combination panels

Uses 96-well microtiter trays/plate Vitek 2 System; Advanced Expert System BD Phoenix

A. ___: Referred to as the gold standard for microbial typing especially those involved in nosocomial infection outbreak. Bacterial chromosome fragments are analyzed to characterize each of the specie’s DNA profile. B. ___: _ targets specific bacteria based on their surface receptors. A culture of bacteria is grown and inoculated with different phages. The positive result is characterized by lysis of colonies which means the specific bacteriophage has successfully attacked the specific bacteria

Pulsed-Field Gel Electrophoresis Bacteriophage typing

__: A DNA sequence that encodes a specific product __: Comprises the all genes in an organism __: Transcription and Translation __: Site of active replication Molecular assay: NEVER USE ___ SWAB with ____ shaft

gene genome Gene expression Replication fork calcium alginate swab; aluminum

POLYMERASE CHAIN REACTION (PCR) invented by __. It involves DNA Amplification or DNA Xeroxy. It is the most common nucleic acid amplification method used in molecular diagnostics. Conventional PCR has __ repetitive thermal cycles: 1. Denaturation (_________) – extract nucleic acid with heat/chemical/enzyme. 2. Annealing (_________) –Primer (specific to target DNA sequence) is added to the denatured DNA. 3. Elongation (_________) – Taq Polymerase is used. Can amplify single copy into 10^7 to 10^8 copies.

Kary Mullis; 20-50 94degC 50-58degC 72degC

-A DNA or RNA segment extracted from the sample which serves as the target for PCR -Oligonucleotide that is specific for the target sequence present in the template -Synthesizes new strands of DNA -AKA RNA-dependent DNA Polymerase, used ONLY in RT-PCR; Not present in other PCR assays. Synthesizes complementary DNA (cDNA) from an RNA template. - Cofactor of DNA Polymerase -Ensures proper pH for DNA Polymerase -Used by DNA polymerase to synthesize new DNA strands during Elongation phase -The instrument used in PCR which adjusts temperature depending on the step being conducted.

Template Primers DNA polymerase Reverse Transcriptase Magnesium Chloride (MgCl2) Buffer Deoxynucleotides (dNTPs) Thermal cycler

Types of PCR: 1. __: Amplification is done followed by gel electrophoresis and band staining to detect amplified products 2. __: (Quantitative PCR or qPCR): Same PCR process of amplification, but accumulation of amplicons is detected real-time by fluorophores that sends fluorescent signals detected by the machine. ~ Reverse Transcription PCR (RT-PCR): __ is used instead of DNA. RNA is first converted into its complementary DNA (cDNA), then usual PCR steps proceed.

Conventional PCR Real-time PCR RNA template

Shaft of Swab: Wooden: Toxic to _____________ and Mycoplasma __: Used usually in a calcium alginate-tipped swab. __: A polystyrene general purpose shaft for bacteria and viruses.

Chlamydia Aluminum Plastic

__ - Generally a poor specimen if aspirate or biopsy can be obtained. APPROPRIATE for nasal/nasopharyngeal, oropharyngeal, eyes, ears, or genitals. NOT FOR __ and __ !!!!

SWABS; ANAEROBES AND FUNGI

Tip of Swab: __: Swabs that are for general use for bacteria and viruses because of excellent collection and release of samples. __: Toxic to ______________________ (unless with charcoal). __: Toxic to enveloped virus & Neisseria, and interferes on immunofluorescent and molecular (PCR) methods

Dacron & Rayon Neisseria Calcium Alginate

Design of Swab: __: Can trap organisms, leading to decreased recovery/sensitivity __: Uses Nylon fibers for enhanced uptake and release of microbes

Woven/Traditional Flocked

Transport specimen in a leak-proof plastic bag with separation for paperwork MARKED with BIOHAZARD label. Most specimen should be transported __ to the lab with some few exceptions which are the sterile specimen (within __). “__” the requirement of microorganisms suspected in a sample.

within 2 hours; 15 minutes Maintain

SHIPMENT: Triple packaging arrangement: ______________________ Sample > Primary container > Absorbent > Secondary container > Dry ice/Liq. nitrogen > Outer/Tertiary container Category A: substances/microbes __ of permanent disability or life-threatening Category B: substances/microbe __ of permanent disability or life-threatening on its current form

Double bagging technique capable incapable

PRESERVATION: * Use __ for URINE (bacteriostatic for 3 days), Don’t use formalin or Polyvinyl alcohol (PVA) - For parasites only * Stuart/Amies may contain _____________ to NEUTRALIZE FATTY ACID that is toxic to bacteria with pili * Anticoagulants are used to prevent clotting of blood, bone marrow, synovial fluid (bacteria will be trapped therefore they’re not going to be isolated).

BORIC ACID charcoal

__ – should be used since some anaerobes and Neisseria are susceptible to increased SPS concentration. can neutralzie with __ __ can also be used for viral culture, and Mycobacterium culture, although it inhibits some gram-positive bacteria and yeasts. __, and others – should never be USED.

0.025% SPS; 1% gelatin Heparin Citrate, EDTA

Abscess, Lesion, Wound, Ulcer Blood: Adults -preferred ___ Pediatric – preferred __ Bone marrow:

BAP, MAC, CAP; Deep abscess – use anaerobic media ≥ 20mL/set; 1-20 mL/set Primary is in Blood culture. Subculture in BAP, MAC, CAP BAP, CAP

Disinfect skin with __ followed by __. (pg 48, Bailey 15E; Ch. 8, Harr) Draw blood during FEBRILE episodes. Draw __. DO NOT draw >4sets in 24hrs period. ARD – Antimicrobial Removal Device Manual - __ blood to broth ratio. Growth detected in the presence of pellicle/turbidity/hemolysis/bubbles. Automated - __ blood to broth ratio. Growth detected by BacT/Alert (5days TAT)

70% alcohol; 2% iodine/iodophor/betadine 2-3 SET only per 24 hours 1:10 1:5

Amniotic, Peritoneal, Pericardial, Pleural fluids Transport within 15 mins at __. CSF/Spinal fluid: In general, Transport: ________ Delayed testing: ______ Storage: __ Viral studies: __ ~__ or use of __ for best sensitivity and organism recovery.

BAP, MAC, CAP, Anaerobic media (Bacteroides) room temperature 22-25degC 20-24degC (Bailey's) 35degC 4degC Cytocentrifugation; acridine orange

Meningitis: Newborn - __ Children - __ Adult - __ Geriatric - __ Immunocompromised (newborns, elders) - __

S. agalactiae, E. coli H. influenzae N. meningitidis S. pneumoniae L. monocytogenes

EAR: Otitis externa: __: S. aureus, S. pyogenes, __ (Swimmer’s ear/Acute diffuse otitis externa) __: P. aeruginosa, Anaerobes Otitis media: __: S. pneumoniae, H. influenzae, M. catarrhalis, S. pyogenes, RSV, Influenza virus, coronavirus, enterovirus, rhinovirus Chronic: __ (Bacteroides, Fusobacterium, Prevotella, Porphyromonas, Peptostreptococcus) __ ear: BAP, MAC, CAP __ ear: BAP, CAP, BBA

Acute: P. aeruginosa Chronic Acute Anaerobes External Inner

Eye conjunctiva or Corneal scraping: Transport within __ at room temp. Corneal scraping is incubated at __ in __.

Direct inoculation to BAP and CAP (transport) 15 mins 28degC; SDA

__Gastric wash or lavage: usually for __. For Mycobacteria detection, process within __ or neutralize with __. Rectal swab: Insert 1-1.5cm deep, feces should be visible on swab. Inoculate in enteric media. ___ - all ages ___ - rotavirus ___ - - >3 days nosocomial diarrhea MEDIA for stool and rectal swab: __ (3); for urine: only ___, ___ URINE (quanti counting): 1uL loop __: 10uL loop ___

infants or AFB 4 hours; 10% sodium carbonate Norwalk – all ages Rotavirus – children C. difficile BAP, MAC, Enteric media; BAP, MAC 1 colony x 1000 = CFU/mL 1 colony x 100 = CFU/mL

Urethra: Swab in __. Collect __ after patient has urinated. culture media: __ __ – N. gonorrhoeae, Chlamydia trachomatis, Ureaplasma, Mycoplasma

Amie’s or Stuart’s medium; 1 hour; Thayer-Martin (culture) Urethritis/Cervicitis

culture medias used for: cervix, vagina and prostate Intrauterine device (IUD): media? Transport within __ at room temp. __ infections.

BAP, Thayer-Martin cervix: +CAP prostate: +MAC, CAP, CNA THIO; 15 mins; Actinomyces

LEVELS OF SPECIMEN PRIORITIZATION: Critical/Invasive specimen Needs immediate processing. Amniotic fluid, Blood, Heart valve, Brain, CSF

Level 1

Nasal swab: culture media? Specimens for viruses, chlamydia, and mycoplasma are usually submitted in appropriate transport media at __ (ice pack or liquid nitrogen).

MRSA agar; MRSA screening 4degC

LEVELS OF SPECIMEN PRIORITIZATION: Unpreserved specimen. Needs to process after Level 1 samples to avoid overgrowth of normal flora or dying of fastidious organisms. Bone, Wound Drainage, Feces, Sputum, Tissue,

Level 2

LEVELS OF SPECIMEN PRIORITIZATION: Quantitation required (1:1000 or 1:100) Catheter tip Urine (midstream/suprapubic/catheterized)

Level 3

LEVELS OF SPECIMEN PRIORITIZATION: Preserved Preserved Urine, Preserved Stool, Swabs in holding/ transport medium

Level 4

Cell wall synthesis inhibitor: Cell membrane synthesis inhibitor: (binds and disrupts) Nucleic acid synthesis inhibitor: Inhibit RNA synthesis - __

Penicillin, Vancomycin, Bacitracin, Carbapenem (penny's vase broken by carl) Colistin & Polymyxin (g-); Daptomycin (for g+) (poor cats die/skibidap) Sulfonamide and Trimethoprim (SXT), Nitrofurantoin, Nalidixic acid, Fluoroquinolones (-floxacin) and Rifampin (sobrang tigas na niya, fuck rara)

Daptomycin, Linezolid, Tedizolid Streptogramin - for __ Colistin & Polymyxin - for __ All gram positive bacteria are __ to Vancomycin except some Enterococcus (VRE), Lactobacillus, Leuconostoc, & Pediococcus. All gram negative bacteria are __ to Vancomycin except some Chryseobacterium, Moraxella, or Acinetobacter.

for G+ only for G- only susceptible resistant

__: Reduced susceptibility of organism to a particular antimicrobial __: Achieved when a particular antimicrobial can no longer be used clinically against a certain bacteria. __: Antimicrobial resistance because of the microorganism’s normal genetic structural or physiological state __: Antimicrobial resistance that results from the “altered” cellular physiology and structure caused by changes in a microorganism’s genetic make-up.

Biologic resistance Clinical resistance Intrinsic resistance Acquired resistance

RESISTANT TO: Anerobic bacteria: Aerobic bacteria: Klebsiella: GN bacteria, Lactobacilli, Leuconostoc:

Aminoglycoside Metronidazole Ampicillin Vancomycin

ANTIMICROBIAL SUSCEPTIBILITY TESTING __ – TT1-TT5 without growth/clear, TT6 –with growth/turbid. This is the lowest concentration of an antimicrobial agent that prevents bacterial growth. __ – broths after incubation are subcultured in agar, number of colonies are counted. This is the lowest concentration of the antimicrobial agent that actually kills the bacteria. __ MacFarland Standard: __ of 1% H2SO4 and __ of 1.175% BaCl2 **A strict full 24 hours incubation is followed if testing for _________ **A strict __ incubator temperature is used if testing for MRSA because __ may mask methR

MIC (Minimum Inhibitory Concentration) MBC (Minimum Bactericidal Concentration) 0.5; 99.5 mL sulfuric acid; 0.5 mL barium chloride VRE, VRSA, MRSA 30-35degC; >35degC

AST: INOCULUM SIZE: Broth Dilution vs Agar Dilution __: REFERENCE METHOD; MIC is determined. Broth microdilution: 0.05 to 0.1 mL broth. Broth microdilution: ≥1 mL broth __: Tests many organisms against 1 antibiotic. Can determine MIC for N. gonorrhoeae which is hard to be grown on broth dilution methods.

BROTH: 5 x 10^5CFU/mL AGAR: 1 x 10^4 CFU/spot broth agar

1.5 x 10^8 CFU/mL Mueller Hinton agar (MHA) is used Add 2% NaCl to detect __; For H. influenzae use __; For N. gonorrhoeae use __: MOST COMMON METHOD; Tests 1 microorganism against __ antibiotic pH media: 7.2 – 7.4; Depth: __ Incubation time: __; 20-24hours if __ or __ Reading a Disk diffusion test: Against a __ background, read with a reflected light (well-lit area). Read zone edges at the point where there’s no obvious growth.

Disk Diffusion/Kirby Bauer MRSA; Haemophilus test medium; GC agar many 4 mm 16-18 hours; Streptococci or Neisseria gonorrhoeae dark

__: The specific concentration of antimicrobial that separate the different categories of susceptibility (Susceptible, Intermediate, or Resistant). __: Antimicrobial agent that is considered the most sensitive indicator of drug mechanism __: A cumulative susceptibility report that tracks resistance or susceptibility of commonly isolated organisms to antimicrobials. __: When there’s HEAVY GROWTH on wells with LOW ANTIMICROBIAL concentration followed by 1 or more wells GREATLY REDUCED GROWTH (small button/light haze). __: When there’s microorganism GROWTH on wells with HIGH ANTIMICROBIAL concentrations and NO GROWTH on 1 or more wells with LOW ANTIMICROBIAL concentrations.

Breakpoint Predictor drug Antibiogram Trailing in broth dilution Skipped wells in broth dilution

KIRBY BAUER: Increase drying Too thin media Use of old cultures (>24h old bacteria) <0.5 MacFarland used Delayed in incubation

FALSE SENSITIVE (large Zone)

KIRBY BAUER: Increase moisture Too thick media >0.5 MacFarland used Delayed application of disks

FALSE RESISTANT (small Zone)

Hazes of Staphylococcus on Methicillin and Enterococci on Vancomycin zones should be read using __ light. __ – ignore swarm, measure the defined zone of inhibition Hazes with sulfonamide & trimethoprim testing – __ the haze, and measure zones If no Proteus/sulfonamide/trimethoprim is used, and hazes are evident, do not ignore and investigate. Plates containing __ is read at the top of the open plate (lid is removed).

transmitted Proteus Swarming Ignore blood

bac resitance: S. aureus, S. lugdunensis 5% Sheep BAP or MHA with 15 μg; ____________ (E) and 2 μg _______(Cd) 35degC, for 16-18 hours bac resistance: S. pneumoniae, beta-hemolytic Streptococci MHA with 5% sheep blood 35degC, 5% CO2, for __ __________________ (Report as Clindamycin Resistant)

D-zone / Inducible clindamycin resistance (ICR) Erythromycin; clindamycin 20-24 hours flattening of clindamycin zone

Modified Hodge test: Enterobacterales __________ production __ like appearance, Indented or Enhanced growth Cefoxitin: to detect ________- mediated oxacillin resistance bac resistance: S. aureus, S. lugdunensis and CONS resistance to __ Disk diffusion using MHA with 30 μg cefoxitin (CNP) test - Confirmatory for Carbapenemase

carbapenemase Clover-leaf mecA oxacillin Carba NP

___ - determines the MIC of 1 antimicrobial against 1 bacteria o _____/Epsilometer – 1 gradient of concentration of antibiotic in a plastic strip o Liofilchem – 1 gradient of concentration of antibiotic in a paper strip __ - is exhibited when a BACTERICIDAL antibiotic can only INHIBIT a bacteria (not killed); most commonly associated to bacterial resistance to beta-lactam antibiotics.

Gradient Diffusion testing E-test Tolerance

__ – AKA Serum-bactericidal test (SBT), activity of drug using patient’s own serum. * __– Bacteria is inoculated in various concentrations of antimicrobial, and incubated, and the plates are subjected to colony counting at different periods of incubation. The rate of killing thebacteria is measured over a specified period (Example: 6 hours, 12 hours, 24 hours incubation) * __ – Identification of the effectiveness of multiple antimicrobial agents against a single bacterial strain.

Schlichter test Time-Kill assay Synergy test

Gram-Positive __ Family: Micrococcaceae Genus: __ Characteristics: Gram-positive cocci, Catalase __, Colonizers

Cocci Staphylococcus, Micrococcus positive

Gram stain: Gram positive cocci in CLUSTERS Aerotolerance: facultative anaerobe Glucose Utilization: fermenter 0.04 U Bacitracin: resistant Furazolidone: sensitive >/= 15mm Modified Oxidase: negative Lysostaphin: sensitive 10-16mm

Staphylococcus

Gram stain: Gram positive cocci in Tetrads/Sarcinae Aerotolerance: aerobic Glucose Utilization: oxidizer 0.04 U Bacitracin: sensitive >/= 10mm Furazolidone: resistant Modified Oxidase: positive Lysostaphin: resistant

Micrococcus

Colonizers of Anterior nares, nasopharynx, perineal area, skin, mucosa Inhibit phagocytosis: Capsule, Slime layer, Biofilm __: Affinity to Fc of IgG and complement Exotoxins: __ – disrupt smooth muscle in blood vessel, toxic to RBC, WBC, platelets, & hepatocyte Beta – work with __ toxin, sphingomyelinase that hydrolyses cell membrane (cell lysis) __ – cytolytic to RBC __ – works with Panton-Valentine Leukocidin; __ toxin __ disease: Scalded Skin, Syndrome/Ritter disease, Pemphigus neonatorum Culture: BAP: Medium-large ____ convex, opaque __, _-hemolytic MSA: __ colonies & halo; CHROMagar: MRSA is __-color; PEA, CNA, MSA: for stool Positive: __ Negative: __ PCR: __ gene (ID of S. aureus), __ gene (ID of MRSA)

Staphylococcus aureus Protein A Alpha alpha Delta Gamma Exfoliatin Exfoliative pinhead smooth creamy yellow β Yellow mauve VP, Slide and Tube, Coagulase!!!, DNase!!! PYR, Microdase nucA, mecA

COCCIS: Inhibit phagocytosis: Slime layer, Biofilm, Delta exotoxin Nosocomial bacteremia from indwelling vascular catheters, endocarditis from prosthetic heart valve, infections from prosthetic joints, etc. Coagulase-Negative!!! (also S. saprophyticus) 5 μg Novobiocin – Sensitive Seen in sexually-active young women urinary tract infection

Staphylococcus epidermidis S. saprophyticus

Tests for Micrococcus and Staphylococcus: Screening test for Gram-positive and differentiates Micrococcaceae from Streptococci (+ is __)? Differentiate Micrococci (+ is color __ in M. luteus) from Staphylococci Differentiate S. aureus from CoNS: 2 types? positive results in S. aureus? Distinguish S. aureus, Serratia, and M. catarrhalis? 2 mediums used? reagents used for each? what is (+) result in S. aureus? (-) result in E. coli?

Catalase test; effervescence Microdase (Modified Oxidase) test; Blue to Purple-blue Coagulase test; tube and slide method SLIDE: clumping; TUBE: clot DNase test; DNase agar and agarose slant; agar: methyl green; slant: toluidine blue and HCl (+) agar: solorless/clearing; slant: deep pink or DNA precipitates (-) agar: green; slant: blue or no ppt

Aids in ID of some GP cocci and bacilli. What is the (+) result (mnemonic) and (-)? Differentiates Clumping factor positive Staphylococci? what is the (+) result for S. intermedius and lugdunensis? in S. aureus?

Voges-Proskauer (Coblentz method) BaBasa Lang Saglit: (RED) B. anthracis, B. cereus, S. aureus, L. monocytogenes, Streptococci anginosus; (-): Yellow PYR test; bright red; no color change

Tube coagulase detects? slide coag? where are they found? temp needed for each? who are the CoNS? (mnemonic)

free coagulase; cell-bound coagulase S: surface of cell T: bacterial suspension S: RT; T: 35degC for 1-4 hours SHEWL: S. saprophyticus, haemolyticus, epidermidis,

Loeffler Serum Slant: Golden yellow Lemon yellow Porcelain white Characteristics: Gram-positive cocci in pairs or chains, Catalase and oxidase negative, Facultative anaerobe Classified according to Rebecca Lancefield grouping except S. pneumoniae and Viridans

S. aureus S. citreus S. albus (S. epidermidis) Streptococcus, Enterococcus, and similar organisms

Streptococcus, Enterococcus, and similar organisms: _____: Pyogenic - Grows at 37degC only; ___ Viridans: Grows at 37degC & 45degC; __ Lactic: Grows at 10degC & 37degC; __ Enterococcus: Grows at 10degC, 37degC, & 45degC; __

Academic/Bergey S. pyogenes, S. agalactiae S. mutans, S. mitis S. lactis E. faecalis

Streptococcus, Enterococcus, and similar organisms: Smith and Brown: Alpha-hemolytic:__ Beta-hemolytic: __ Gamma-hemolytic: __

S. pneumoniae S. pyogenes, S. agalactiae S. bovis

Streptococcus, Enterococcus, and similar organisms: Lancefield: (mnemonic) Group A: Group B: Groups C, G: Group D:

PADEE S. pyogenes S. agalactiae S. dysgalactiae, S. equi Enterococci

(Group A) Streptococcus pyogenes: ___– Responsible for 2 sequelae and binds Factor H; can cause what? ___– Epithelial cell attachment (Fibronectin binding) DNase; Streptokinase; Hyaluronidase; ___ – Scarlet fever Taxo A: __; PYR: __; ASO titer; ___; Schulz-Charlton test STREP O vs S:

M protein; Rheumatic fever (Class I) & Acute Glomerulonephritis (Class I or II) Protein F Pyrogenic exotoxin Sensitive; Positive; Dick’s test O: Oxygen labile, Antigenic, Subsurface hemolysis (stab) S: Oxygen Stable, Non-Antigenic, Surface hemolysis (streak)

GROUP AND SPECIES? VF: Capsule, Activation of complement LIM, Todd-Hewitt, Granada, Carrot medium VF: Endogenous strain of skin, nasopharynx, GI, and genital that may gain access to sterile site; β-Hemolytic VF: Biofilms, Adhesins (medical implants), Cytolysin, Hemolysin, peptide inhibitors Nosocomial or Healthcare-associated; Enterococcosel agar, Positive in Bile esculin, 6.5% NaCl tolerance, and PYRase

(Group B/GBS) Streptococcus agalactiae (Group C) Streptococcus dysgalactiae subsp. equisimilis and other group A, C, G, L (Group D) Enterococcus faecalis Enterococcus faecium

Normal flora of nasopharynx; VF: Capsule – Pneumolysin!!!, mediate oxidative outburst in phagocytosis Secretory IgA protease Phosphorylcholine – Spreading and invasion Causes: Meningitis, CA-Pneumonia, Otitis media, Microscopic : Encapsulated __ shaped diplococci Macroscopic: __-hemolytic, mucoidal, __ colonies __ Sensitive, Bile solubility, Neufeld quellung

Streptococcus pneumoniae bullet/lancet Alpha; umbilicate Optochin *(bullet sa head, kita umbilical cord, alpha male, may suka na green sa chin)*

Normal flora of oral cavity, GI tract, female genitals Alpha hemolytic, __ smell on CAP, __ positive (pink color), PYR negative

Viridans Streptococci Butterscotch; LAP

Tests for Streptococcus: Presumptive test for S. pyogenes: (2) Presumptive test for Enterococci and Streptococcus bovis; Organisms are capable of GROWING in __ and hydrolyzing it to esculetin; Reagent: Ferric ammonium citrate (Iron); (+) ___ ppt Presumptive test for S. pneumoniae, differentiates it from other alpha-hemolytic Streptococci? reagent for the BST is 10% or 2% Sodium deoxycholate (+) result on S. pneumoniae For Enterococci; reagent: Cinnamaldehyde; (+) result on E. faecalis? (-) result on Aerococcus viridans

Bacitracin susceptibility (BAP) and PYR test (+) is bright red Bile Esculin Hydrolysis; 40% bile; growth AND blackening (negative if one only) Optochin (P disk) susceptibility test and Bile solubility test; Colony in agar disintegrates or Broth culture becomes clear. LAP test; Red; Yellow/No color change

Tests for Streptococcus: Differentiate Group B streptococcus from other Streptococci; (+) result on S. agalactiae? (-) result on S. pyogenes? Presumptive ID of some organisms; reagent: 0.2 mL ninhydrin reagent; (+) result on S. agalactiae; (-) result on S. pyogenes Tests for Enterococcus, faecalis (+) vs Enterococcus faecium; reagent: Bromothymol blue; (+) result on E. faecalis; (-) result on S. gallolyticus For Enterococci's ability to withstand high salinity; reagent: Bromocresol purple; (+) result on E. faecalis; (-) result on S. gallolyticus

CAMP test, arrowhead hemolysis; no enhanced/arrowhead hemolysis Hippurate hydrolysis; deep purple; colorless or yellow pink Pyruvate test; Yellow; No change or Yellow-Green/Weak Salt Tolerance test; Growth & Yellow; Little/no growth

General characteristics: Gram positive bacilli, Aerobic, Spore-forming Category A Bioterrorism Soil-isolate contracted by animals (Exposed industrial raw worker, military, public health personnel) VF: Capsule; TOXINS: __ and __; toxins are made of __ as its factor DSE: Cutaneous anthrax (or___): Pulmonary/Inhalational anthrax (__): Gastrointestinal or Ingestional anthrax (_): Injectional anthrax: Gray, Flat, __, comet-tail/__ or ground glass colonies in BAP; Large Gram positive bacilli in chains resembling __ Spore-inducing techniques: Growth in TSI, Urea, NA with Manganese sulfate, or bicarbonate medium Heat shock (degC and mins?) Selective media: __; Presumptive test: 10 U Penicillin susceptibility test (__ appearance), PCR (__ capsule gene)

Bacillus spp. Bacillus anthracis; Lethal toxin (LT), Edema toxin (ET); Protective antigen (PA); Black eschar; Woolsorter’s or Rag-picker’s disease: Toxemia and sepsis. Nonhemolytic, Medusa-head, bamboo-square 62-65degC for 15-20mins. PLET agar; String of pearl; capBCA

Blood bank contaminant: VF: Emetic type is Heat-stable, acid- resistant Cereulide enterotoxin Diarrheal type is Hemolysin BL (HBL), Nonhemolytic enterotoxin (Nhe), Cytotoxin K (CyK AKA hemolysin IV) Large, feathery, spreading __ on BAP, Wide zone of clearing in __________; Culture media: MEYP/MYP, PEMBA, BCM (anything egg agar); Heat shock: 70degC for 30mins. or 80degC for 10mins. Ingestion of toxins in rehydrated foods; same appearance on BAP with B. cereus; toxins are: Cytotoxin _, enterotoxin

Bacillus cereus Beta hemolytic; egg yolk agar Bacillus cytotoxicus; K

Cereulide toxin is used in insecticides and pesticides: __ eye infection among drug users: __ B. subtilis is large, ___, ___, ground- glass, __-hemolytic, and may be pink/yellow/orange pigmented colonies on BAP B. thuringiensis – Large, __, spreading __ hemolytic on BAP; Parasporal crystals/Insecticidal proteins Large, moist blister colony with ameboid spreading in young cultures while wrinkled old cultures that is non-hemolytic seen in BAP

B. thuringiensis B. subtilis flat, dull; beta feathery; Beta Paenibacillus polymyxa

B. anthacis vs B. cereus: wide-zone lecithinase, capsule, motility, hemolysis, gelatinase, penicillin G?

B. anth: +, +, -, -, - sensitive B. cer: +, -, +, +, +, resistant

# ___: General characteristics: Gram positive bacilli, Non-spore forming bacilli __ are short-slightly curved rods with rounded ends arranged in palisades Not part of Normal flora. Nasopharynx in carriers. VF: Diphtheria toxin (inhibits protein synth) Biotypes: 1. Intermedius – _______, gray, translucent β-hemolytic colony 2. Mitis – ______-sized, convex, smooth, black pigment fried-egg appearance, β-hemolytic colony 3. Belfanti – medium-sized, grayish-black, opaque, β-hemolytic colony 4. Gravis – ______, flat, glossy, grayish-black, daisy-head appearance, __-hemolytic colony STRAINS: toxigenic is respiratory diphtheria; non-toxigenic is Cutaneous diphtheria or Velat sore/Barcoo rot Presumptive: Brown-Black colonies with gray-brown __ in __ agar Nutrients: Cystine; Culture: Tinsdale, __ medium (No ↑CO2) Toxigenic strain ID: _______immunodiffusion test, Tissue culture, EIA, PCR, Schick skin test, Roemer test (Guinea pig virulence test)

Corynebacterium spp., Listeria spp., Erysipelothrix spp., and the likes Corynebacterium Corynebacterium diphtheriae small; medium; large; Non halo; Tinsdale; Loeffler; ELEK;

Zoonosis: cattle (Summer outbreak); Urease & Reverse CAMP (+); Glycogen fermentation (+), Zoonosis: sheep, goats, horses; causes Suppurative granulomatous lymphadenitis; Urease & Reverse CAMP (+); Glycogen fermentation (-), Colonizer of Male genitourinary tract; Positive CAMP, glucose and sucrose fermentation

Corynebacterium ulcerans Corynebacterium pseudotuberculosis Corynebacterium glucoronolyticum

Normal flora of skin of hospitalized patients in inguinal, axillary, or rectal sites or from Intravenous catheters; Multiple antibiotic resistance in hospital setting; seen in indwelling catheters, prosthetic heart valves, CSF shunts Enhanced growth with lipids (lipophilic): Add __ or Serum in media; __ on Nitrate and Urease Normal flora of skin; same as above in medium; Struvite crystals in urine, Rapid urease + Normal flora of skin; causes ___ or superficial, chronic, pruritic reddish-brown macules in skin. Lesions are __ fluorescence under a Wood lamp

Corynebacterium jekeium; Tween 80; Negative Corynebacterium urealyticum Corynebacterium minutissimum; Erythrasma; coral red

Colonizers in animals, soil, vegetable. Sources: Meat, processed foods, soft cheese, vegetables, dairy products like milk, or colonized mother to fetus. VF: __ - pore-forming toxin, hemolytic, cytotoxin that reduces responsiveness of T-cells; __ – Eats iron from transferrin; __ – Escape WBC and spread in blood; __ – placental invasion; __ – induce phagocytosis; __ – causes actin polymerization resulting to pseudopod projections causing cell-to-cell infection DSE: Neonatal Listeriosis, Granulomatosis infantisepticum Short gram-positive bacilli that may occur in singly or short chains __ motility when incubated at NB at RT for 1-2 hours. ___ growth or ___ in SIM at 22-25degC. __ enrichment: Specimen in 4oC for weeks-months Selective media: PAL-CAM, McBride medium; __ test - purulent conjunctivitis in rabbit; __ in Catalase, CAMP (__-type hemolysis), Voges- Proskauer, Esculin; __ on H2S, Urease

Listeria monocytogenes Listeriolysin O; Siderophores; Phospholipase; E-cadherin; Internalin; Act A Tumbling; umbrella-like; inverted Christmas tree; Cold Anton; Positive; Block Negative

Zoonosis of veterinarians, meat (pork) butchers, and fish handlers; Normal flora VF: Capsule, Neuraminidase, Hyaluronidase, Surface proteins DSE: ___, Bacteremia Gram positive short bacilli forms long filaments __-hemolytic on bap __ on catalase, oxidase, esculin, nitrate, urease; __ in TSI. Test-tube brush/Pipe cleaner appearance in gelatin stab culture at 22degC Curved Gram-positive rods with pointed ends and rudimentary branching, Reverse CAMP positive Normal flora of vagina, GI tract; Pleomorphic (Long chaining bacilli, coccobacilli, spiral forms); __-hemolytic on BAP

Erysipelothrix rhusiopathiae erysipeloid Alpha; Negative; (+) H2S; Arcanobacterium haemolyticum Lactobacillus acidophilus; mouthAlpha

Actinomcyetes WITH Mycolic acid on Cell wall; Acid-fast (underlined): Partial acid-fast (not underlined):

Nocardia, Rhodococcus, Gordonia, Tsukamurella, some Corynebacterium Lawsonella, Segniliparus

Cell wall peptidoglycan has Diaminopimelic acid (DAP), Arabinose, and Galactose; In agar: Substrate hyphae and Aerial hyphae __ appearance, with hyphae. Partial Acid Fast, __, __ & __ positive. LYSOZYME Resistant; Extensive Hyphae In __. Culture media: SDA, LJ, BHI, BCYE, MTM – Incubate at __ for 2-3 weeks at __. Sulfur Granules from specimen emulsified in 10% KOH, crushed, and Gram stained: Small yellow-orange __ shaped with __-like structure __ – most common Nocardia isolate. NEGATIVE CASEIN, Xanthine, Tyrosine Hydrolysis __: positive in Casein & TYROSINE Hydrolysis

Nocardia spp. Beaded; Strictly aerobic; Catalase & Urease; Tap Water Agar 30degC; ambient air; kidney; club Nocardia asteroides (sensu stricto) Nocardia brasiliensis

Mucoid orange to red or Salmon-pink non-hemolytic colonies Gram positive coccobacilli with “zigzag” scanty branching pattern Positive in Urease, Catalase, & CAMP

Rhodococcus equi

Glabrous Waxy heaped gray-white colonies Gram positive atypical bacilli with “spore-like bodies” and extensive branching that do not fragment Musty basement odor Extensive Hyphae In Tap Water Agar - LYSOZYME Sensitive

Streptomyces spp.

Gram positive actinomycete Gold standard testing: PAS stain in gastrointestinal biopsy First identified via PCR of duodenum biopsy causes: __

Tropheryma whipplei Whipple’s disease

General characteristics: Acid-fast bacilli (with Mycolic acid), Aerobic, Agents of pulmonary and skin diseases __ is seen mostly in the poor, homeless, IV drug users, alcoholics, & the elderly. __ – Productive cough, hemoptysis, Low-grade fever, night sweats, anorexia, weight loss. In CXR, formation of Gohn lesion with “granulomas/hard tubercles”, giant cells (cellular fusion with multiple nuclei). Spread is through lymphatic or blood causing Meningeal/Miliary/Disseminated tuberculosis (common in immunocompromised). __ - precedes with no S&S, no active disease, and not transmissible. Pott’s disease or tuberculosis spondylitis/skeletal tuberculosis – __

Mycobacterium spp. Tuberculosis Pulmonary TB bone TB

Biosafety Class: __: Acid fast smears and culture for NON-aerosolizing procedures. __: Manipulation with Aerosolizing procedures, Opening centrifuge vial, adding reagents to biochemical tests, molecular tests, and sonication. After aerosolizing procedures, stand specimen for 15 minutes.

Biosafety Level 2 Biosafety level 3

M. tuberculosis vs M. bovis? Niacin Nitrate reduction Pyrazinamidase Catalase Urease T2H/TCH Specimen: Sputum (DOH, __ samples; Bailey __ samples – each are 8 hours apart), __: (specimen of choice for <12 years old), 3 consecutive First-morning midstream clean catch urine, Stool (AIDS, M. avium complex), Tissue samples (incubated at 35oC and 30oC), SPS, Heparin, or Citrate Blood culture, and CSF.

M. tub: positive all; catalase neg; resistant to T2H/TCH M. bov: negative all; T2H/TCH senstive 2; 3 Gastric lavage

Digestion-Decontamination methods (FOR TB): 1. __ – 2-4% NaOH, both decontaminant and digestant. 2. Zephiran-Trisodium phosphate method 3. __ – most commonly used for TB specimen processing; used in MGIT 960. Other reagents used in TB: 1% Cetylpyridinium chloride – prolongs shelf life if sputum sample for up to __ __ – Decontaminant for thin samples like Urine. __ – used for patients with cystic fibrosis (M. abscessus) __ specimen: Centrifuge and use sediment for microscopy and culture __ specimen: Liquefaction → Decontamination → Neutralization → Centrifugation → use sediment for microscopy and culture

NaOH method. NALC-NaOH method 8 days Sulfuric acid Chlorhexidine Sterile Nonsterile

Culture media for TB: A. Solid Media 1. Egg-based/Opaque media: __ and __; Lowenstein-Jensen, American Thoracic Society (ATS) Medium, Petragnani medium, Wallenstein medium. MYCOBACTERIUM TUBERCULOSIS COMPLEX 2. Serum-based/Transparent media: __ 7H10 and 7H11 and __ 7H11. __ is for protection of AFB against toxins. Casein hydrolysate for Isoniazid-resistant MTB B. Liquid Media 1. BACTEC 12B: used for Mycobacteria Growth Indicator Tube (MGIT 960) system 2. Middlebrook 7H9 broth and Dubos Tween Albumin broth C. Biphasic Media __ AFB – mixture of TB culture media for rapid identification of mycobacteria Automated methods for Mycobacteria 1. BACTEC 460 TB System: 2. Mycobacteria Growth Indicator Tube (MGIT 960) system: 3. BacT/Alert MB 4. versaTREK culture

has Egg yolk and Malachite green Middlebrook; Mitchison; Albumin SeptiCheK

Immunologic Tests for TB: uses skin hypersensitivity to induce erythema and induration; Poor sensitivity, & anergy on patients with HIV. MEDICATIONS: __/__ Anti-Tubercular drugs: Rifampicin, Isoniazid, Pyrazinamide, Ethambutol __: Resistant to at least two first line anti-TB drugs. __: Resistant to Isoniazid, rifampicin, fluoroquinolones, and at least one of the 5 second-line TB drugs (Amikacin, Amithiozones, Bedaquiline, Ethionamide, or Capreomycin).

Mantoux/Tuberculin/PPD First-line/Primary MDR-TB XDR-TB

Nontuberculous Mycobacteria characteristics: Runyon Group? Classification: Photochromogens, Scotochromogens, Non-Photochromogens. what are their growth rate? color when dark or exposed to light Rapid Growers' Runyon grp? growth rate?

phot: GROUP I scot: GROUP II non: GROUP III 10-21 days; Cream/Buff for dark, Yellow to Orange after light exp GROUP IV; 3-7 days

Nontuberculous Mycobacteria: GROUP I Fresh water and Salt water; Fish with contaminated water or breaks on skin; __ Tween 80 Hydrolysis, Urease, Pyrazinamide DSE: Cutaneous disease (__), bacteremia; Optimum growth temperature: __ In tropical environmental water (primarily in Australia); DSE: Pulmonary disease, bursitis, tenosynovitis Found in tap water, More common in white Males __, Catalase, Tellurite Reduction, & Tween 80 Hydrolysis DSE: Chronic Pulmonary disease, cervical lymphadenitis, cutaneous disease

M. marinum; Positive; Swimming pool granuloma; 28-32degC M. asiaticum M. kansasii; positive in nitrate M. branderi other species in GRP I: M. intermedium and M. nebrakense

Nontuberculous Mycobacteria: GROUP II Fresh/Tap water (pipeline, laboratory faucet) Positive Catalase and Tween 80 hydrolysis; Raw milk, dairy products, soil; causes cervical adenitis in children contaminants in water and soil

M. gordonae M. scrofulaceum M. szulgai other species under GRP II: M. europaeum, M. interjectum, M. heckeshornense & M. lentiflavum

Nontuberculous Mycobacteria: GROUP III Environmental isolate; Positive Tellurite reduction and Pyrazinamidase; DSE: causes Johne disease in cattle, sheep, goats; Patients with AIDS: Disseminated disease Immunocompromised: coinfection with HIV Disseminated disease in AIDS patients Hot water taps in hospitals; Optimum growth temperature: 28-32oC Stagnant topical waters, aquatic insects; Positive heat-stable catalase; can cause African Buruli ulcer or Australian Bairnsdale ulcer

M. avium complex (MAC) M. celatum M. genavense M. xenopi M. ulcerans other species in GRP III: M. shimoidei, M. simiae, M. terrae complex, M. lacus, M. malmoense & M. haemophilum

Nontuberculous Mycobacteria: GROUP IV __% pulmonary disease caused by this group; it is cystic fibrosis-associated seen in environmental, Water, soil, dust

M. abscessus; 80%; M. fortuitum and M. chelonae

RGT: 30% H2O2 and 10% Tween 80 (+): >45mm of bubbles (M. flavescens) (-): <45 mm column of bubbles (M. tuberculosis) RGT: same as above + water bath (+): Bubble formation (M. flavescens) (-): No change (M. tuberculosis) RGT: Nutrient broth with 1% Potassium nitrate; Indicator: Sulfanilic acid (+): red (M. tuberculosis); (-): No color change (M. marinum) RGT: Lowenstein Jensen with ≥50 colonies; (+): Yellow (M. tuberculosis); (-): No color change (M. fortuitum) RGT: Tween 80; Indicator: Phenol red (+): Pink color in 10 days (M. kansasii); (-) No color change (M. intracellulare)

Semi-Quantitative Catalase test (SQC) 68degC Heat-stable Catalase test Nitrate Reduction Niacin accumulation Tween 80 hydrolysis

RGT: Culture media with T2H; (+) No growth (Sensitive, M. bovis); (-) Growth (Resistant, M. tuberculosis) RGT: Ferric ammonium sulfate and Pyrazinoic acid; (+): Red/Pink band in 4 days (M. intracellulare); (-): Colorless/Gray (M. bovis) RGT: Iron-uptake test 20% ferric ammonium citrate; (+): Rusty brown colonies in egg-based media (M. smegmatis and M. fortuitum) (-) No color change (the rest of Mycobacteria) RGT: Urea broth; (+): Pink/Red in 3 days (M. tuberculosis); (-): No color change (M. gordonae) RGT: 5% NaCl added to egg-based media; (+) Substantial Growth (M. fortuitum); (-): No growth(M. gordonae)

T2H Inhibition Pyrazinamidase test Iron-uptake test Urease test Salt tolerance test

Causes Leprosy (Hansen disease) __ phase: Multiplication in skin macrophage __ phase: Multiplication in peripheral nerves __ form: Tuberculoid leprosy Disseminated form: Lepromatous leprosy (Facial and nasal deformities, Lepromin skin test negative) Laboratory methods: ____ appearance under the microscope NOTCULTIVABLE in regular media (in vitro) but Cultivable in ___ or mice (in vivo). Phenolase test positive; Fite faraco stain

Mycobacterium leprae Silent Intermediate Localized ciagrette packet/picket fence foot pads of armadillo

General characteristics: Catalase and Oxidase positive, Gram-negative diplococci, Capnophilic (5-10% CO2). ___ are fastidious/fragile organisms that are temperature-dependent and are inhibited by __ and increased SPS concentrations (0.025% lang dapat). Hold cultures for up __ in CO2 incubator before reporting as negative/no growth. Other Neisseria spp. are environmental isolates and has low virulence and associated to opportunistic infections.

Gram-Negative Cocci Neisseria; fatty acid 72 hrs

NEISSERIA T1 and T2 are virulent strains; Reduced recovery: >30 mins. delay, use of cotton tip or calcium alginate swab, oil-based lubricant. MOT: sexual contact or verrtiacl transmission VF: Pili (T1-T5), Capsule, Endotoxin, Protein II (Opa), Protein II (RMP), Porin (PorB); can cause Perihepatitis (Fits-High-Curtis) PCR/NAAT specimens: Endocervical/urethral swab or urine __-bean or __-bean shaped diplococci inside PMN. Selective media: ___ agar; Culture media: CAP, __ (3) AST media: gonoccocus agar (GC agar) ~ 4+ in __; CTA: __ only

Neisseria gonorrhoeae Kidney; coffee GC-LECT Superoxol test (30% H2O2); glucose

NEISSERIA: MOT: from respiratory droplets, close contact VF: IgA protease, Proteins like Por and Opa; can cause Waterhouse friderichsen syndrome Specimen: CSF, Blood, NPS, skin; Culture media: BAP, CAP, TMA ~ Catalase and Oxidase +, Beta-galactosidase and GGT + CTA: __ and __ only (CTA (+) is yellow, (-) is pink)

Neisseria meningitidis glucose and maltose

NEISSERIA: Small and translucent colonies with yellow pigment CTA: ___?; Lactose fermenter, ONPG (+) dry, wribkled colonies exhibiting bread crumbs Grows on BAP and CAP at 25degC; CTA: __ Colonizers of upper respiratory tract and female genitalia. Transmission through respiratory droplets; __, __ are responsible for beta-lactamase production; ___________________ (intact colonies) or old “Wagon wheel” appearance, __ , Beta-lactamase +, Butyrate+, Can grow on Nutrient agar

Neisseria lactamica; Lactose, Glucose, Maltose Neisseria sicca: Sucrose, Glucose, Maltose, Fructose Moraxella catarrhalis; BRO-1, BRO-2; hockey puck colonies; DNAse +

Rapid ID of M. catarrhalis; (+) result: ___ (M. catarrhalis); (-): No color change (__) ID of N. meningitidis: (+) result: __; (-) No color change (M. catarrhalis)

Butyrate/Tributyrin test; Blue/Indigo; N. gonorrhoeae GGT or GGAP; yellow

Carbohydrate utilization medium: __; Reagent: 1% carbohydrate; Indicator: Phenol Red Incubate at 35-37degC for 24-72 hours in ambient air. Positive: __ color. Negative control: ___ CTA tube.

Cystine Trypticase Soy Agar; Yellow; Uninoculated

Oxidase negative, Gram-Negative Bacilli: Enterobacterales - Gram negative __ bacilli (not curved) - Opportunistic pathogens except for Overt/True Pathogens: E. coli (Diarrheagenic), Salmonella, Shigella, Plesiomonas, and Yersinia - All are motile except ___ (mnemonic) ~ though who is motile at room temp 25degC? - ALL ARE OXIDASE (-) except for ___ - All are catalase + Nitrate reduction + Glucose fermenter

long slender straight SKY (Shigella, Klebsiella, Yersinia); Yersinia Plesiomonas

Rapid Lactose fermenters? mnemonic Late Lactose Fermenter? Non-Lactose Fermenter?

EcEK: Escherichia coli, Enterobacter, Klebsiella SeCSea: Serratia, Citrobacter, Salmonella enterica subsp. aizonae; ( ShYPMMS: Salmonella, Shigella, Yersinia, Hafnia, PMP group

Tests for Gram-Negative (Enterobacterales): Aid in ID of Gram-negatives along with Gram stain; RGT: Longwave UV light; (+) Blue fluorescence (E. coli); (-) No fluorescence (S. aureus) Differentiates Enterobacterales from other GN bacilli; (+) Dark purple/Deep Blue (P. aeruginosa); (-) No color change (E. coli) For Enterobacterales members (all +); (+) RED after addition of indicator. (E. coli); (-) Colorless or Change of color after zinc dust (Acinetobacter baumanii) Further Enterobacteria test; RGT: Solution A: Sulfanilic acid and Solution B: a-naphthylamine (both reductions); (+) Colorless, gas production (P. mirabilis); (-) Red, no gas (Acinetobacter baumanii) Tests bacteria for their oxidative or fermentative ability; RGT: Phenol Red; (+) Yellow (acids) Oxidizer: P. aeruginosa, Fermenter: E. coli; (-) none

Gram Sure Oxidase test (Kovac’s method) Nitrate Reduction Nitrite Reduction Oxidation and Fermentation (CDC Method)

Kauffman-White classification; Serological identification of Enterobacterales spp.: __: Somatic antigen, Cell wall, Heat-stable, E. coli and Shigella serotyping __: Capsular antigen, Capsule, heat-labile; Detection of __ Antigen of E. coli and capsular __ antigen (Virulence) of S. typhi __: Flagellar antigen, Flagella; Heat-labile; Heat-labile

O antigen K antigen; K1; Vi H antigen

Tests for Gram-Negative (Enterobacterales): For decarboxylase - producing Enterobacteria; RGT: Bromocresol purple and Cresol red; Lysine → Ornithine → Arginine → Purple: Lysine – K. pneumoniae Ornithine – K. aerogenes Arginine – P. aeruginosa Yellow: Lysine – C. freundii Ornithine – P. vulgaris Arginine – E. coli

Moeller’s Decarboxylase test Cadaverine; Putrescine; Citrulline (LOA:CPC)

Tests for Gram-Negative (Enterobacterales): For Enterobacterales; Aerobic Slant: __; Anaerobic Butt: __; RGT: Bromocresol Purple and Ferric ammonium citrate (H2S) Lysine decarboxylase → Lysine deamination → Glucose fermentation → H2S produced → Lysine decarboxylation: (K/K) - Klebsiella, E. coli, Edwardsiella, Salmonella, Serratia Lysine deamination and glucose fermentation: (R/A) or R/Y: __ group: Quality Control: K/K +H2S: C. freundii K/A: __ R/A: Proteus mirabilis

LIA Lysine; Glucose Purple slant (K) Red slant (R) Yellow butt (A) black precipitate PMP E. coli

Tests for Gram-Negative (Enterobacterales):For Enterobacterales; Aerobic Slant: Lactose,(__), Sucrose (__), Anaerobic Butt: Glucose (__ ); RGT: Phenol Red (acid production) and Ferric ammonium citrate (H2S) RXN: Ⓐ - Acid/Acid + gas Ⓐ - K/A, g/G, H2S+ - Salmonella enterica subsp. enterica serovar Typhimurium K/A, H2S+ - __ K/A – __

Triple Iron Sugar 10%; 10%; 1% E. coli P. mirabilis Shigella flexneri

Tests for Gram-Negative (Enterobacterales): A: Differentiate enteric bacteria from Coryneforms; B: Differentiate Enterococci from Streptococci RGT: (A) Andrade’s indicator & Durham tube; (B) Bromocresol purple. A: (+) Pink With gas (E. coli), Without gas (S. flexneri); (-) Clear or straw, no gas (Coryneforms) B: (+) Yellow with gas (E. coli); (-) Pink, no gas (Moraxella osloensis) Positive for Moraxella lacunata, Serratia orodifera, Proteus spp., Pseudomonas; RGT: Refrigeration; (+) Partial (top layer) or total liquefaction of tube at 4degC (Bacillus subtilis); (-) Complete solidification of tube at 4oC (E. coli) Distinguishes late lactose fermenter from non-lactose fermenters; (+) Yellow (Shigella sonnei); (-) Colorless (Salmonella enterica serovar Typhimurium)

Fermentation media Gelatin Hydrolysis ONPG test (o-Nitrophenyl-β-D-Galactopyranoside)

Tests for Gram-Negative (Enterobacterales): For IMViC reactions; positive for PMP group, E. coli, Klebsiella oxytoca Plesiomonas, Edwardsiella, Citrobacter koseri (PEKPEC); RGT: Kovac’s reagent (dimethylamine-benzaldehyde & HCl), Ehrlich’s reagent (__ and __ are added) (+) Pink/Wine color RING after addition of indicator; Colorless/Yellow Positive Red: Citrobacter, E. coli, PMP, Salmonella, Shigella (CEPSS); RGT: Methyl Red; (+) RED (E.coli); (-) YELLOW (K. aerogenes) (+) Klebsiella, Enterobacter, Serratia, Hafnia (KESH); RGT: Solution A: ɑ-naphthol, Solution B: KOH; (+) RED (K. aerogenes); (-) YELLOW (E.coli) For IMViC reactions; MED: Simmons citrate agar slant; RGT: Bromothymol blue; (+) Growth; Blue (K. aerogenes); (-) Small/No growth; Green (E. coli) Differentiate E. coli from Shigella; Ability to use the Sodium acetate as sole source of carbon; RGT: Bromothymol blue; (+) Growth; Blue (E. coli); (-) Small/No growth; Green (S. sonnei)

Spot Indole Ehrlich’s reagent Methyl Red Voges-Proskauer (Barritt’s method) Citrate utilization Acetate Utilization

Tests for Gram-Negative (Enterobacterales): Presumptive ID of Proteus spp. and other enterobacteria; RGT: Phenol red; (+) Magenta/Red (P. vulgaris) Other positive organisms: (Klebsiella, PMP group, Nocardia, Rhodococcus, Helicobacter, Ureaplasma, Cryptococcus) (-) Light Orange/Yellow (E. coli) Detects motile organisms; Motile bacteria produces diffuse zone of growth; (+) Spreading (P. vulgaris); Colonies remain on site of stab (K. pneumoniae) Detection of EHEC/E.coli O157:H7 (no fluorescence); RGT: 366 nm Ultraviolet light; (+) Electric Blue fluorescence All E. coli except O157:H7; (-) No fluorescence (K. pneumoniae) and E. coli O155:H7

Urease (Christensen’s method) Motility test MUG test

Tests for Gram-Negative (Enterobacterales): Detects PMP/PPM group (Positive); RGT: 10% FeCl3; (+) GREEN (P. mirabilis); (-) Yellow (E. coli) Rapid detection of Vibrio spp.; __ lyses DNA of VIBRIO only, forming a string; (+) String-like between loop and slide (Vibrio spp.); (-) No string formation

PAD test String test; 0.5% Sodium deoxycholate

#1 cause of community-acquired and hospital-acquired UTI, most common gram-negative healthcare-associated infections, and gram negative sepsis #2 cause of neonatal meningitis (k1 antigen + E. coli)

Escherichia coli

Pili heat stable (ST); heat labile toxin; Cholera-like toxin (water and electrolyte secretion) DSE: #1 in Traveler’s diarrhea, Childhood diarrhea, Montezuma’s revenge Profuse Watery Diarrhea (Cholera-like diarrhea) Strains: E. coli O4, O8, O25 Oversecretion of cyclic adenosine monoPO4 (cAMP)

ETEC or Enterotoxigenic E. coli

Pathogenicity island virulence factors: Bundle of pili, intimin, toxins: loss of microvilli DSE: Infantile diarrhea, Mucoidal diarrhea, Can be in __ Strains: E. coli O55, O111, O114 Seen in infants in low-income nations

EPEC or Enteropathogenic E. coli Infant formula

Invades large intestine in a manner identical to Shigella. Invasin toxin, Large plasmid, (Sereny test +) DSE: Dysentery (necrosis, inflammation, and ulceration of large intestine) Shigella-like manifestations Strains: E. coli O124, O143, O164 Seen in young children in areas with poor-sanitation

EIEC or Enteroinvasive E. coli

Shiga-like toxin Verotoxin/Verocytotoxin DSE: Bloody diarrhea, H.U.S. (hemolytic uremic syndrome), Hemorrhagic colitis Strains: E. coli O157:H7, O26 Undercooked hamburger, unpasteurized dairy products, apple cider, bean sprouts, spinach, cookie dough

EHEC / VTEC / STEC or Enterohemorrhagic E. coli

__ – clumped/aggregates in intestine through pili; watery diarrhea __ – diffuse patterns on HeLa or Hep2 cells, causes diarrhea mostly in children __ - #1 cause uncomplicated UTI __ – K1 Antigen positive

Enteroaggregative E. coli (EAEC) Diffusely-adherent E. coli (DAEC) Uropathogenic E. coli (UPEC) Meningitis/sepsis-associated E.coli (MNEC)

Ingestion of contaminated poultry products (milk, eggs, dairy) All Salmonella are __ except Salmonella serotype Gallinarum and Typhi All Salmonella produce H2S except __. Salmonella serotype Typhi and Paratyphi persists in __ (gall stones). Cholecystectomy – Tx for chronic carriers Salmonella sampling: 1st week: __ 2nd week: __ 3rd week: __

Salmonella spp. motile Salmonella serotype Paratyphi A; gall bladder blood stool urine

Salmonella: __ (Typhoid fever) - Contaminated food from food handlers that are carriers (Typhoid Mary) Long term carrier: __ Commonly caused by S. enterica subsp. enterica serotype Typhi Malaise, anorexia, myalgia, severe frontal headache Complications: __ Hallmark: __ during second week of fever __ (Food poisoning) Commonly caused by S. enterica subsp. enterica Salmonella Typhimurium outbreak (contaminated __ cracker and cereals); Nausea, vomiting, fever, chills, watery diarrhea, abdominal pain

Enteric fever gallbladder Necrotizing cholecystitis Rose spots Acute Gastroenteritis; peanut butter

Biochemically inert bacteria; Closely related to E. coli Infections caused by contaminations from Four F’s (?) Shigella that produces __ weakens agglutination for serotyping. Suspend isolate in saline and heat in waterbath for 15-30 mins at __. Cool then retype with antisera.

Shigella spp. Flies, Fingers, Food, Fecal capsule; 100degC

SHIGELLA: Group A; Most virulent (Shiga toxin); DSE: Bacillary dysentery NEGATIVE for ONPG, Ornithine Decarboxylase and Mannitol fermentation

Shigella dysenteriae

SHIGELLA : Group B; Pathogenic, common isolate; DSE: GAY BOWEL SYNDROME!!!; NEGATIVE for ONPG & Ornithine Decarboxylase, mannitol ferm +

Shigella flexneri

SHIGELLA: Group C; Pathogenic, less incidence; DSE: Dysentery; NEGATIVE for ONPG & Ornithine Decarboxylase, mannitol ferm +

Shigella boydii

Clinically Relevant Enterobacterales spp./Other Pathogenic Enterobacteria: Non-motile, Urease negative; Class A bioterrorism agent; Vector: Oriental rat flea, Reservoir: Rats; DSE: Bubonic/Glandular plaque & Pulmonary plaque: Optimum growth temperature: __degC; Bipolar staining/__; __ pattern in broth Motile at 25degC or RT; Urease positive, Ornithine and Sucrose positive; MOT: thru undercooked pork, pork intestine, vacuum-packed meat and chicken, dairy & pets; DSE: Waterborne-Enterocolitis & Appendicitis-like syndrome; Cold enrichment Bull’s eye colonies in __ Peritrichous flagella; in BB: __ contamination Motile at Room Temp; Urease positive, Ornithine and Sucrose negative; Agent of rodents, Reservoir: Birds; __ colonies with transparent borders in CIN Pathogenic; Ingestion of contaminated seafood, oyster, shrimps, water; MOT: Entry on Skin cuts; The ONLY oxidase POSITIVE!!! member of Enterobacteriaceae IMViC: ___ TSI: __

Yersinia pestis; 25-30degC; Safety-pin appearance; Stalactite Yersinia enterocolitica subsp. enterocolitica; CIN; Packed RBC Yersinia pseudotuberculosis; Red pin-like Plesiomonas shigelloides; +,+,-,-; K/A H2S(-)

SHIGELLA: Group D; Self-limiting, most common isolate; DSE: Dysentery; POSTIVE for ONPG & Ornithine Decarboxylase and mannitol ferm

Shigella sonnei

Other Non-Pathogenic/Opportunistic Enterobacteria: Food and fish/water contamination; LDC positive, Non-lactose fermenter, Same IMViC to E. coli: ___; TSI: __ __ (red) pigment at 25degC; Bacteremia outbreaks in nurseries, cardiac surgery, and burn units. Swarming; Antigens of __ - OX19, OX2; Antigen of __ - OXK; DSE: Acute glomerulonephritis, Urinary tract infection (UTI), Kidney stone formers ~ PAD positive, Urease positive, LDA positive: (5)

Edwardsiella tarda; (+,+,-,-); K/A g+ H2S(+) Serratia marcescens; Prodigiosin Proteus; Proteus vulgaris; Proteus mirabilis ~ Proteus, Morganella morganii, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii

Currant jelly sputum, Capsule (Mucoid), Some are Carbapenemase producer; Large pink mucoidal colonies in MAC, ___ positive, __ positive Blue safety pin appearance; DSE: Granuloma inguinale or Donovanosis; Donovan bodies in endothelial cells Can break sialic acid in breast milk and infant formula. Contaminant of powdered infant formula!!!!!; IMViC: __ Nosocomial outbreaks due to contaminated IV fluids; Negative TSI reactions but positive glucose fermentation (K/A) May be mistaken as Salmonella in biochemical reaction (Citrobacter is LF); Differentiate with indole or H2S

Klebsiella pneumoniae, potassium cyanide (KCN), String test Klebsiella (Calymmabacterium) granulomatis Cronobacter (Enterobacter) sakazakii; -,-,+,+ Pantoea (Enterobacter) agglomerans Citrobacter freundii (positive for H2S) & Citrobacter koseri (C. diversus) (positive for indole)

ENTEROBACTERALES DIFFERENTIAL TESTS + COLONY CHARS: A/A g K/K + + – – – MAC: pink/Red SSA: Pink/Red XLD: Yellow HEA: orange to salmon pink K/A g K/K – – + + – MAC: Pink(Late) SSA: Colorless XLD: Colorless HEA: Colorless A/A G K/A – – + + – MAC: pink/Red SSA: Pink/Red XLD: Yellow HEA: orange to salmon pink A/A G K/K – – + + + MAC: pink/Red SSA: Pink/Red XLD: Yellow HEA: orange to salmon pink

Escherichia coli Serratia marcescens Enterobacter cloacae Klebsiella pneumoniae

ENTEROBACTERALES DIFFERENTIAL TESTS + COLONY CHARS: A/A G K/K + – + + + MAC: pink/Red SSA: Pink/Red XLD: Yellow HEA: orange to salmon pink A/A G K/K – – + + – MAC: pink/Red SSA: Pink/Red XLD: Yellow HEA: orange to salmon pink A/A g K/A + + – + – MAC: Pink/Red SSA: Pink/Red XLD: Yellow HEA: orange to salmon pink A/A H2S g K/A – + – + – MAC: Pink/Red SSA: P/R (H2S) XLD: Y (H2S) HEA: Y (H2S) Salmonella enteritidis K/A H2S g K/K – + – + – MAC: Colorless SSA: C. (H2S) XLD: R/C.( H2S) HEA: G/C.(H2S) MAC: SSA: XLD: HEA:

Klebsiella oxytoca Enterobacter aerogenes Citrobacter diversus Citrobacter freundii Salmonella enteritidis

ENTEROBACTERALES DIFFERENTIAL TESTS + COLONY CHARS: K/A H2S v R/A + + – + + MAC: Colorless SSA: Colorless (H2S) XLD: R/C (H2S) HEA: G/C (H2S) K/A H2S g R/A – + – + + MAC: Colorless SSA: Colorless (H2S) XLD: R/C (H2S) HEA: G/C (H2S) K/A H2S K/K – + – – – MAC: Colorless SSA: C. (H2S) XLD: R/C.( H2S) HEA: G/C.( H2S) K/A K/A – + – – – MAC: Colorless SSA: Colorless XLD: Red/C HEA: Green/C K/A K/A – -v – – – MAC: Colorless SSA: Colorless XLD: Red/C HEA: Green/C

Proteus vulgaris Proteus mirabilis Salmonella serotype Typhi Shigella dysenteriae & Shigella sonnei Shigella flexneri & Shigella boydii

K/A H2S g K/K + + – – – MAC: Colorless SSA: C. (H2S) XLD: R/C.( H2S) HEA: G/C.( H2S) K/A R/A + + – + + MAC: Colorless SSA: Colorless XLD: Red Or Colorless HEA:Red Or Colorless K/A R/A + + – + – MAC: Colorless SSA: Colorless XLD: Red Or Colorless HEA:Red Or Colorless K/A g R/A + + – – + MAC: Colorless SSA: Colorless XLD: Red Or Colorless HEA:Red Or Colorless K/A K/A v + – – + MAC: Colorless SSA: Colorless XLD: Red Or Colorless HEA:Red Or Colorless

Edwardsiella tarda Providencia rettgeri Providencia alcalifaciens Morganella morganii Yersinia enterocolitica

Curved, Oxidase positive, Gram-Negative Bacilli: General characteristics: Agents of gastroenteritis, Catalase Positive, Oxidase Positive Genus: (4) __ – Gram negative Curved/Comma-Shaped Bacilli, Catalase, Oxidase, Indole, Glucose Positive, Motile via __, Facultative Anaerobe; Found in ___; __ except V. cholera and V. mimicus; Infections: Cholera, Septicemia, Wound infections

Vibrio, Campylobacter, Arcobacter, Helicobacter. Vibrio spp.; Polar flagella; Salt/Brackish/Marine water; Halophilic

Pandemic strain: VC-O1 Biotypes: __ - VP +, B-hemolytic, agglutinates chicken RBC, polymyxin B resistant; Classical Serotypes: Ogawa, Inaba, Hikojima VF: Choleragen, Cholera toxin (CT), Zonula occludens toxin (Zot), Accessory cholera toxin (Ace), Flagella, Mucinase, Toxin Coregulated Pili (TCP); DSE: Cholera HALLMARK: __; Transport media: Cary-Blair *NO TO GLYCEROL!!* Enrichment media: pH 8.4; agar used? Culture media: pH 8.6; agar used? String test positive, Vibriostatic test (0129-impregnated disk) sensitive

Vibrio cholerae; El Tor; rice watery stool APW; Alkaline peptone water; TCBS

V. cholerae (Cholera; Rice water stool) V. alginolyticus (Otitis, wound infection, gastroenteritis)\ V. furnissii V. fluvialis V. cincinnatiensis V. metschnikovii

Sucrose Fermenter ; Yellow in TCBS

Non-Sucrose Fermenter ; Green in TCBS causes Summer diarrhea, B-hemolysis in Wagatsuma agar causes Septicemia, wound infection, Lactose + V. mimicus

V. parahemolyticus V. vulnificus

Aquatic environments, Freshwater, polluted, chlorinated, brackish water, occasionally marine water DSE: Human: Gastroenteritis, wound infection, bacteremia Reptile: ___ Gram negative, straight bacilli with rounded ends, Oxidase positive __ colonies on CIN __-hemolytic on BAP, Do not grow on TCBS., __ , grows with 0% NaCl

Aeromonas spp. Red-leg syndrome Bull’s eye, Beta NOT halophilic

Gram-negative Curved/Seagull wing-shaped bacilli Catalase, Oxidase, _______ Positive; Microaerophilic. Grows on 42degC (Hot enrichment method); DSE: Associated with gastroenteritis, reactive arthritis (Reiter syndrome) and ___ Improved staining: 0.1% basic fuchsin Culture media: Skirrow, CAMPY-CVA, Cefoperazone agar, Charcoal-based selective medium (CSM) DSE: Causes gastroenteritis and septicemia; __ method enhance recovery of Campylobacter and __ in stool samples using 0.45 to 0.65 um pore size polycarbonate or cellulose acetate filters; Plate is __ for 1 hour at 37oC. ~ __ gram negative bacilli that are Oxidase positive, __ Catalase positive, & Urease NEGATIVE

Campylobacter spp. indoxyl acetate; Guillain-Barre syndrome Arcobacter spp.; Filtration; Arcobacter; upright Curved; weak

DSE: Causes Peptic Ulcer & Peptic Cancer Genetic Virulence: - __ produces exotoxin that creates vacuoles and loosens tight junctions - __ is a part of Pathogenicity islands causing peptic carcinoma - __ binds Leb antigens in gastric epithelial cells - __ causes peptic ulcer Other Helicobacter: H. rappini, H. cinaedi, H. fenneliae – isolated from bacteremic homosexual men with HIV; ~ Catalase, Oxidase, Motility, Urease Positive!!! (Urea Breath Test) Microaerophilic, Curved Gram negative bacilli; Tissue biopsy transport media: Brucella broth with glycerol, Stuart’s transport medium, Portagerm pylori media Stain: ___ (3)

Helicobacter pylori VacA CagA BabA IceA 0.1% basic Fuchsin, Warthin-starry (silver), Giemsa

Oxidase positive, Gram-Negative Bacilli General characteristics: Aerobic, Non-spore forming, & Motile with __ (except Burkholderia mallei) Genus: Pseudomonas, Burkholderia, Cupriavidus, Ralstonia, Agent of Nosocomial infection, Infection of wound (Blue pus), Burn and Diabetic patients, Cystic Fibrosis and COPD patients, Dermatitis in __ and __, Ecthyma gangrenosum – black and necrotic skin papules, ___ AKA Malignant Otitis externa, __ solution contamination ~Catalase, Oxidase +, Acetamide +, Cetrimide growth +, Growth at __ __ odor (2-Acetaminophenone); Virulence factors: __ (kill host cell through protein synthesis inhibition), Elastase, Phospholipase C, Pili, ___ (disrupt cytoskeleton) Pigments: __, (Pyorubin, Pyoverdin, Pyomelanin) Biofilm producer (Alginate) – inhibitor of phagocytosis ___ – detects bacterial products in environment

polar flagella Pseudomonas aeruginosa; hot tubs and Jacuzzi; Swimmer’s ear infection; Contact lens 42degC, Grape-like; Exotoxin A; Exoenzyme S and T Pyocyanin Quorum sensing

Infection on patients with cystic fibrosis: __ Plant pathogen that is isolated from on patients with cystic fibrosis and chronic granulomatous disease (CGD): ___ Burkholderia mallei: Causes severe infection in horses (_____ disease) Burkholderia pseudomallei or __; Melioidosis/Glander’s-like disease AKA the _____ or Great Mimicker; Sample: __ (3) ; Dry, Violet-purple, Wrinkled colony on __ Common in environment; Brown/yellow Adherent Wrinkled colonies Nosocomial/catheter-related infections. ______. Beta-lactamase +. LDC, DNase and Motility positive. ___ in BAP. __ odor

Burkholderia cepacia Glander's disease (Whitmore’s bacillus); Vietnamese time bomb; Throat swab, Rectal swab, Sputum; Ashdown's agar Pseudomonas stutzeri Stenotrophomonas maltophilia; oxidase negative; Lavender green; Ammonia

Drug resistant. Nosocomial infection. An ESKAPE pathogen. ________ Catalase + __ in MAC. __ of carbohydrates (A. lwofii is not an oxidizer) __ coccobacilli, resist decolorization Causes Conjunctivitis; Gelatinase +. Liquefy/Digests ___ slant. Causes neonatal meningitis; Old genus: Flavobacterium, Chryseobacterium; Produce __ pigment; Oxidase and DNase + Associated to chronic granulomatous disease (CGD), sepsis, and necrotizing fasciitis; Violacein pigment. Almond odor/____ odor.

Acinetobacter baumanii; oxidaaaaase negative (dami a); Lavender; Oxidizer; Plump Moraxella lacunata; Loeffler’s serum Elizabethkingia meningoseptica; flavin/Yellow. Chromobacterium violaceum; ammonium cyanide

ESKAPE GROUP – group of highly virulent, antibiotic-resistant, nosocomial infection-associated bacteria. Small, nonmotile, capnophilic, pleomorphic Gram negative bacilli or coccobacilli; Fastidious/fragile organisms requiring __ and __. Capsule is composed of Sugar-alcohol (polyribitol) phosphate; __ capsular serotype: H. influenzae type a to f __ – Normal Flora, “Satellite Phenomenon”; Meningitis, Epiglottitis, Cellulitis, Arthritis, Pneumonia; Otitis Media: transmitted through __. Particle agglutination for CSF and urine. __ - Brazilian purpuric fever __ – Acute otitis media, sinusitis, bacteremia, culture negative endocarditis __ – purulent conjunctivitis, pink eye __ – Chancroid/Soft chancre (genital lesions → papules → painful ulcers), a STD; School of red fish

Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterobacter spp. Haemophilus spp.; X and V factors; 6 H. influenzae; respiratory droplets H. influenzae biotype aegypticus H. parainfluenzae H. aegypticus H. ducreyi

HAEMOPHILUS SPP: group that needs X and V factor but dont produce porphyrin group that needs V factor and produce porphyrin group that needs X factor ONLY group that only produces poprphyrin

H. influenzae, H. aegypticus, H. haemolyticus (1st row) H. parainfluenzae, H.parahaemolyticus, H. paraphrophilus (PiPhaPaP or p- pop) [2nd row] H. ducreyi {3rd row} Aggregatibacter aphrophilus

To differentiate Haemophilus spp. Media: Heart infusion agar, TSA, NA, Haemophilus medium Reagent: __ impregnated with ___ factor; Incubate: Overnight, 35oC, ambient air Positive results: Growth around XV disk only: 1st row of the table Growth around XV and V disks only: 2nd row of the table Growth around XV and X disks only: 3rd row of the table Negative result: Growth around XV, X, and V disks: __

X and V factor test Disk/strip; X, V, & XV Non-fastidious organisms

* 5 dysgonic small gram negative (cocco) bacilli * Normal oral flora but occasionally associated with Subacute Bacterial Endocarditis!!!! * Recovered in blood culture then Subculture in BAP/CAP (Candle jar incubation) A: seen in Dental plaques, X factor dependent, Nitrate reduction + A: seen in Periodontitis; Coinfection with Actinomyces; Star shaped centers in BHIA C: Frequently associated with aortic valve infection; The only Indole positive in AACEK!!!!! False gram positive staining - ___; Rosette (rose petal) formation with stick-like projections under microscope E: Human bite infection Bleach-like odor, ______ K: Joint and bone infections; osteoarthritis in children Narrow zone of beta hemolysis in BAP

AACEK (HACEK): Aggregatibacter (Haemophilus) aphrophilus Aggregatibacter (Actinobacillus) actinomycetemcomitans Cardiobacterium hominis; pits agar Eikenella corrodens; pits agar Kingella kingae

CATALASE, OXIDASE +; BEST Specimen: Nasopharyngeal swab (NPS) Virulence: Pili; Causes __ cough/100 days fever (Catarrhal, Paroxysmal, Convalescent stage); Prevented with DPT vaccine Media: Potato Blood glycerol/Bordet Gengou agar, Regan Lowe/Charcoal Cephalexin Blood agar, Casamino broth, Jones Kendrich

Bordetella pertussis; whooping

Bordetella: - the only oxidase positive!! - grows on BAP and uerase positive - positive in Motility, Nitrate reduction, Oxidase, Urease, Growth

B. pertussis B. parapertussis B. bronchiseptica

Thionine and Fuchsin inhibition tests is used for: only specie inhibited by thionine is: __ species inhibited by fuchsin is: __ not inhibited by both is: __ DSE: Animal and Human pathogen ___, Undulant fever, __, ___, Mediterranean fever, Animal abortion; Found in animal placenta/erythritol. Transmission: Ingestion of Unpasteurized or contaminated milk/cheese, Inhalation through animal carcasses, needle-stick/skin abrasions Common lab-acquired infection through aerosols

Brucella spp. B. abortus B. suis, B. canis B. melitensis Brucellosis; Bangs disease; Gibraltar disease

GRAM NEGATIVES: Deerfly fever, Rabbit fever; Require Cysteine & Cystine. __ skin test. Glucose Cysteine Blood agar, Peptone Cysteine agar. Shipping fever. Bioterrorism. Cat-Bite fever!!! Oxidase, Catalase, Glucose, Indole +;_______ odor Cat-Scratch disease (#1)!!! Oxidase, Catalase negative. Warthin Starry stain. causes Cat-scratch disease Dog-bite infection, Periodontal disease; Gliding motility, Fusiform shaped Gram negative; Sensitive to SPS Rat-Bite fever/Sodoku!!!; dse Helix shaped. Sensitive to SPS Oroya fever, Verruga peruana/__, Carrion’s disease!!!; History. Vector: __

Francisella tularensis; Forshay Pasteurella multocida; mushroom Bartonella henselae Afipia felis Capnocytophaga spp. Spirillum minor/minus Bartonella bacilliformis; Peruvian warts; Sandfly

GRAM NEGATIVES: Haverhill fever; Broth: __ and fluff balls appearance; Agar: __ appearance with dark center and __; Stained with __ for Microscopy Broadstreet pneumonia AKA __. Pontiac fever (no pneumonia); Faint in Gram stain, use __ or 10 mins. Safranin. From Air conditioning unit, condensers, humidifiers, nebulizers, & cooling towers. DFA to detect antigens in urine, respiratory samples, & tissues. Normal flora of vagina; Bacterial vaginosis; Nugent scoring system: __; __ –10% KOH __ odor; Gram-variable or gram-negative coccobacilli and short bacilli, Catalase neg, Oxidase neg, Inhibited by __; Hippurate positive, __ Primary Atypical Pneumonia or __, Pleuropneumonia-like organism (PPLO), Stevens-Johnson syndrome; Confirmed using HEMADSORPTION (using guinea pig RBC) from Edward Hayflicks medium. Use SP4 media with GLUCOSE or NYC agar; Post-abortal, post-partum fever; HIV-associated cervicitis; __, embedded in media. Use SP4 media with __, NGU, PID, Prostatitis, Infertility, Sexually acquired arthritis, Enhances HIV transmission; Glucose metabolism Positive, __ metabolism and Urease Negative Urethritis (NGU), neonatal infection; Use SP4 media with UREA or NYC agar

Streptobacillus moniliformis; Breadcrumbs; Fried egg; lacy edges; Dienes Legionella pneumophila; Legionnaire’s disease; Fuchsin Gardnerella vaginalis; Clue cells; Whiff/Sniff test; Fishy-amine odor; SPS; HBT agar Mycoplasma pneumonia; Walking pneumonia Mycoplasma hominis; Mulberry/Fried egg colonies; ARGININE Mycoplasma genitalium; Arginine Ureaplasma urealyticum

ANAEROBIC BACTERIOLOGY: __ percent Oxygen requirement – ___ (throat, NPS, rectal, etc.); Holding temperature: ROOM TEMP (20-25oC). Culture media: PRAS media, Reduced media, Roberston’s Cooked/__ media, Thioglycollate broth, Anaerobic blood agar, AnaeroPak system, Gas Pak Jar system, Brewer jar, McIntosh and Filde’s jar, Boiling Indicator of Anaerobiosis: ___

ZERO; NO SWAB Chopped meat Resazurin and Methylene blue (BLUE)

Indicators of the Prescence of Anaerobic Bacteria 1. __ odor 2. __ granules 3. __ fluorescence on UV light 4. __ of hemolysis 5. Lack of __

Foul, Sulfur, Brick red, double zone; Superoxide dismutase

Obligate intracellular Nonculturable Bacteria: Energy parasites/Obligate Intracellular __ are like gram-negative in a way that they have LPS as well in their cell wall. Antigenic variations is due to __; __ body – Metabolically Inert, Infective form; __ body – Intracellular, Active Replicating form

Chlamydia spp. Chlamydia; MOMP (Major Outer Membrane Protein); Elementary; Reticulate

ANAEROBES: Gas gangrene bacillus; VF: Encapsulated, Central to Subterminal spores (Histotoxin)!!!! __-shaped appearance; Double zone of hemolysis, __ positive on EYA; Nagler test +; __ +; DSE: Gas gangrene, Necrosis, __ disease Tack-Head bacillus; ____spore, Tetanospasmin (Neurotoxin); Drumstick/lollipop/Racquet appearance; Swarming on BAP, Lecithinase negative; DSE: Tetanus, Lock jaw Trismus, __, muscular rigidity and __ Caned good bacillus; _____spore (Neurotoxin); With __ spore resembling tennis racket; __ positive on EYA ___ bioassay – Identification of Botulinum toxin (BoNT); DSE: Foodborne botulism, Botulism, __ syndrome, __ (rag doll) paralysis; blurred vision, slurred speech, dysphagia ________: gold standard for detection of toxin Toxin A (TcdA) – __; Toxin B (TcdB) – __; Toxin C and binary toxin; Rods in chains up to __. With __ spores; __ on CCFA; __ odor on BAP;DSE: Pseudomembranous colitis

C. perfringens; Boxcar; Lecithinase ; Reverse CAMP; Pig-bel C. tetani; terminal spore; Risus sardonicus; spasm C. botulinum; subterminal; Lipase; mouse; Floppy baby; Flaccid C. difficile; tissue/cell culture; enterotoxin; cytotoxin; 6; oval; Yellow ground glass; Horse manure

Obligate intracellular Nonculturable Bacteria: #1 Ocular Trachoma (Serovars A, B, C) #1 Pelvic Inflammatory disease (Serovars D to K) #1 Lymphogranuloma venereum (Serovars L1, L2, L3) #1 Non-Gonococcal urethritis #1 STD in __ TRachoma and Inclusion Conjunctivitis agent __ syndrome TESTS: Use __ shafts of swab (Endocervix, Vaginal, Urethral) Best Media: __ Growth enhancer: __ Best Test: PCR/NAAT Test Chlamydia antibody: Microimmunofluorescence inclusions: __ bodies; Glycogen-containing bodies; Stain: __

Chlamydia trachomatis U.S.; Fits-Hugh-Curtis plastic/metal; McCoy Cell Line; Cycloheximide Halberstadler-Prowazeik; Lugol’s iodine

Obligate intracellular Nonculturable Bacteria: Psittacosis, Ornithosis, Parrot fever, Pneumonia (__ cough); -from inhalation of bacteria in __ Sample: Sputum, NPS, OPS Media: ___ Microimmunofluorescence and PCR inclusions: __ bodies; Non-Glycogen containing stain: __ and __

Chlamydophila psittaci; Hacking; bird’s dropping/feces; Mice or chick embryo Levinthal-Lillicole Macchiavello and Giemsa

Obligate intracellular Nonculturable Bacteria: TWAR strain; Taiwanese Acute Respiratory Syndrome Ass. To asthma and atherosclerosis Sample: Sputum, NPS, OPS, __ Media: HeLa or Hep-2 Microimmunofluorescence and PCR stain: Giemsa

Chlamydophila pneumoniae; bronchial wash

TWAR strain of pneumonia Currant jelly-like sputum Rust colored sputum; #1 community acquired pneumonia Primary Atypical Pneumonia (PAP) Broadstreet pneumonia

Chlamydophila pneumoniae Klebsiella pneumoniae Streptococcus pneumoniae Mycoplasma pneumoniae Legionella pneumoniae

Cell wall is like a Gram-negative cell wall. Obligate intracellular and non-cultivable in cell-free media; Attachment to host cell - ____ Infection occurs after a bite of an infected __ vector; Test is usually: NAAT (PCR) or Serology (e.g. Weil-Felix)

Rickettsia; Omp A & Omp B; arthropod

Spotted Fever group: African tick bite fever Mediterranean/Israeli/Boutonneuse spotted fever; Indian/Kenya tick typhus Far-Eastern tick-borne rickettsiosis Flinders Island spotted fever Japanese spotted fever Rocky Mountain spotted fever Mild illness Rickettsia sibirica North Asia tick typhus Tick-borne lymphadenopathy

Rickettsia africae Rickettsia conoriii Rickettsia heliongjiangesis Rickettsia honei Rickettsia japonica Rickettsia rickettsi Rickettsia parkeri Rickettsia parkeri Rickettsia sibirica Rickettsia slovaca vector: Ticks

Typhus group: Epidemic typhus; vector: Lice Brill-Zinsser disease (Recrudescent disease); vector: None Murine typhus; vector: Fleas Scrub typhus group: Scrub typhus; vector: Chiggers Others: Human MONOCYTIC; vector: ehrlichiosis Ticks (Ambylomma spp., Lone star tick) Human GRANULOCYTIC naplasmosis; vector: Ticks (Ixodes spp.) Sennetsu fever; vector: Ticks

Rickettsia prowazekii Rickettsia typhi ~~ Orientia tsutsugamushi ~~ Ehrlichia chaffeensis Anaplasma Neorickettsia sennetsu

Is a gram negative coccobacillus which causes Q (Query) fever!!!! and can be Isolated from animals. Survives extracellularly and can only be grown in __ cells. No rash development. Phase I (Large-cell variant): Phase II (Small-cell variant): Acting like a __, extracellular (in the environment) Zoonotic infection with cattle, sheep, & goats Biosafety level ____ Tests: Shell vial culture using Human lung fibroblast (1 day) Serology - IFA - PCR -

Coxiella burnetti; lung spore 3 most common test reference method more sensitive test than sero

General characteristics: Long, slender, __ curved __ organism Examined under __ microscope __ – platelet-like structure in the cell wall in which fibrils are attached Axial fibrils/Axial filaments/Endoflagella – flagella-like organelles wrapped around the bacterial cell wall Genus and their morphology: - __ (Tight coils) - __ (Thicker, Loose coil) - __ (Thicker, Loose coil, Hooked ends) - __ (Comma-shaped/Helical, Tapered ends with 4 flagella on each end)

Spirochetes; helically; microaerophilic; dark-field; Insertion disks Treponema Borrelia Leptospira Brachyspira (spiral)

Hutchinson’s triad: ______, _____, Notched peg-shaped teeth; Bone malformations: Bulldog maxilla, Saber shin (tibia); Blood or CSF X-ray, Immunologic test

Congenital syphilis; deafness, blindess

SYPHILIS STAGES: Primary syphilis: __ Chancre in genitals; SPX: Lesion Swab/Aspirate; TEST: Dark-Field microscope, Culture Secondary syphilis: __ rashes in palms and soles; SPX: Lesion aspirate; TEST: Sero Latent syphilis: __ phase; No signs/symptoms;SPX: Serum; TEST: Serology Tertiary syphilis: Tissue-destruction; Granuloma-like lesions (__). Cardiac and CNS problems, ___ SPX: Lesion. Blood. CSF. Serum. Tissues; TEST: Serology, PCR – Neurosyphilis in AIDS patients

Hunterian chancre/Hard Condylomata lata Subclinical Gummas; Neurosyphilis

Relapsing fever: ____ - stained Blood smear Blood and CSF (1st week); Urine (2nd week) Culture: Fletcher, Noguchi, EMJH media Gold standard-__ Blood (Acridine orange or Giemsa), biopsy (Warthin-Starry stain), CSF, or Urine; Culture: Chick embryo, Barbour-Stoenner-Kelly media; Confirmatory – __ Reaction from teteracycline treatment of spirochetes

Giemsa Leptospirosis; Microscopic agglutination (MIT) Lyme’s disease; Western blot Jarisch Herxheimer reaction

Species of Spirochetes: Syphilis Yaws or Fambresia tropica Bejel or Endemic Non-venereal syphilis Pinta or Carate Gingivitis, Periodontitis Louse-borne/Epidemic/European relapsing fever Tick-borne/Endemic/American relapsing fever Lyme’s disease!!!! Leptospirosis / Infectious Jaundice / Weil’s disease!!!

Treponema pallidum subsp. pallidum Treponema pallidum subsp. pertenue Treponema pallidum subsp. endemicum Treponema carateum Treponema denticola and Treponema socranski Borrelia recurrentis Borrelia hermsii, B. turicatae, B. duttoni, B. parkeri Borrelia burgdorferi Leptospira interrogans

Stages of Lyme’s disease: Erythema chronicum migrans, Fever, malaise, joint pain Second stage Neurologic disorders (nerve palsy), meningitis Acrodermatitis chronica atrophicans (recurring chronic arthritis) & Demyelination symptoms of Alzheimer’s disease and Multiple sclerosis

First stage Second stage Third stage

QUALITY CONTROL IN MICROBIOLOGY: Laboratory goals of __: * Improving the accuracy of patient identification * Improving the effectiveness of communication among caregivers * Reducing the risk of healthcare-associated infections. __: is associated with the internal activities that ensure diagnostic test accuracy. It is defined as the measures designed to ensure the medical reliability of laboratory data. __: is associated with the external activities that ensure positive patient outcomes. __: concentrates on eliminating redundant motion, recognizing waste, and identifying what creates value from the client’s perspective __: is a concept introduced by Motorola in 1986 that reduces defects by minimizing variation in processes through metrics measurement. Methodology involves DMAIC __: Defined as the accuracy, reliability, and timeliness of reported test results.

The Joint Commission (TJC) Quality control Quality Assurance LEAN Six Sigma Laboratory quality

International Organization for Standardization (ISO): __ - Medical laboratories requirements for quality and competence. __ - General requirements for the competence of testing and calibration laboratories. __ - General requirements for the competence of providers of proficiency testing schemes

ISO 15189:2007 ISO/IEC 17025:2005 ISO/ IEC 17043:2010

Classification of errors in AST reporting: * __: adequate interpretation of the susceptibility test result. * __: Microorganism shows intermediate sensitivity to a given antimicrobial but is reported as susceptible or resistant; microorganism is susceptible or resistant but reported as showing intermediate resistance. * __: classification as resistant of a susceptible strain (false resistance). * __: classification as susceptible of a resistant strain (false susceptibility). In-use/working culture: 1 vial per week is thawed and _______

No errors in interpretation Minor error (Mi) Major error (Ma) Very major error (Vma) subcultured twice

Lab-related Government Agencies and Function: - Oversight of public health and safety including the laboratory - Regulation and Approval of in-vitro reagent kits - Calibration of thermometer, weights and measures; International system of Units - Culture media quality control; Format of ____ Standardization of laboratory practices - Control organisms used for AST (20 days testing) - Licensing of laboratories - Test kit or method validation, Proficiency testing; Bioterrorism investigation and confirmation along with CDC Provides Research, education, and training in the field of occupational safety and health; Certification of respirator masks (e.g. N95)

CDC (Centers for Disease Control and Prevention) FDA (Food and Drug Administration) NIST (National Institute of Standards and Technology) CLSI (Clinical and Laboratory Standards Institute); SOP manuals ATCC (American Type Culture Collection) DOH (Department of Health) RITM (Research Institute for Tropical Medicine) NIOSH (National Institute of Occupational Safety and Health)

RITM NEQAS Frequency: Bacteriology Parasitology Mycobacteriology Microscopy and Culture - Transfusion Transmissible infections (HIV, HBV, HCV, Syphilis, Malaria): __ SARS-CoV-2 Molecular

Once a year - all except TTIs Twice a year - TTIs only

Record/Specimen type Retention: Urine: __ Microbiology stained slides: __ Body fluid and Smears: __ Test Requisitions: __ Patient records: __ Culture media performance and sterility records: __ QC records: __ Retired Laboratory procedures: __ Maintenance of records with billing concerns: __ QC record of instrument: __

urine: 24 hrs slides, body fluid and smears: 7 days test req-retired: 2 yrs maintenance: 10 years qc record: As long as instrument is being used

DIAGNOSTIC WATER BACTERIOLOGY ___– index of fecal contamination in a drinking water. __ – indicator organisms of contamination in water. __ parameters to be tested (DOH): E. coli/coliform, Arsenic, Cadmium, Lead, Nitrate, Color, Turbidity, pH, total dissolved solids, and disinfectant residue. Bacteriologic analysis of water: (1) Total plate count; (2) Presence/absence of coliform; (3) MPN count Methods of Microbiologic Water analysis: Multiple tube fermentation (MPN count), Membrane filtration, Defined substrate, Presence/absence of coliform, Pour plate method, Chromogenic substance

E. coli coliforms 10 Mandatory water

_DIAGNOSTIC WATER BACTERIOLOGY: _____ – presence of residual chlorine indicates there’s sufficient amount of chlorine to inactivate bacteria/viruses and that water is protected from recontamination!!!! during storage. Residual chlorine should be __ 30mins. after addition of NaOCl and __ 24 hours after addition of NaOCl in water. ______ – neutralizes free chlorine in swimming pool water. NOTES: Incubation of broth/cultures are mostly 24-48 hours at 35oC (others were up to 44.5oC to detect thermotolerant E. coli). Positive results include _____ in broths and __ colonies in plated media. Indicators commonly used are __ (for acid production, yellow) and __ (for gas detection, bubble).

Chlorine residual testing; 2 mg/L; 0.2 mg/L sodium thiosulfate acid and gas production; characteristic coliform!!!!; phenol red; durham tube

- Quantitative (Reported in Most Probable Number → the number of coliform resulting from multiple-portion decimal dilution plantings/ determines number of coliform /checks for the potability of water) -Gold standard is __ of test tubes. MPN is __ from a standard chart based on no. of tubes with positive result. __: >1.1 MPN/100 mL __: <1.1 MPN/100 mL

Multiple Tube Fermentation test (MTFT) 5 set; calculated Positive Negative

Stage 1: __ > Lauryl Tryptose broth / Lactose broth + water sample (Incubate at 35oC for 24-48hrs) → Acid and Gas production (+) Stage 2: __ > To confirm __, use __ medium, otherwise for __ use __. > Brilliant green lactose bile broth (BGLBB) or E. coli (EC) broth + inoculum from stage1 → gas production+!!! (NRL manual) > EMB / Endo agar + inoculum (from stage1) (Incubate at 35oC for 24-48hrs) → colonies (Observe green metallic sheen!!!) (Conventional methods in U.S.) Stage 3: __ > Single strength Lauryl Tryptose broth + inoculum → Gas production (+) and LES Endo agar or MacConkey agar look for gram negative bacilli, then perform traditional IMViC for differentiation of coliforms. (DOH, NRL manual) > Lactose broth + inoculum (from stage 2) Incubate at 35oC for 24-48hrs) → look for acid and gas production (Conventional methods in U.S.)

Presumptive test Confirmatory test TOTAL COLIFORM; BGLBB FECAL COLIFORM; EC broth Completed test

___ (Formerly: Standard Plate count) Negative: <500 CFU/mL (Dialysis water <200 CFU/mL) -estimates the number of live heterotrophic bacteria in water; measures changes during water treatment and in swimming pool -Indirect indication of pathogen removal from water treatment infacilities. METHODS: Pour plate method, Spread plate method, and Membrane filter method. CULTURE MEDIA: R2A, NWRI agar, PCA __ – detects coliforms from the environment, food, and beverage (Incubation is 35degC for 24 hours; characteristic color forms in the presence of coliform bacteria).

Heterotrophic Plate count Millipore water sampler

MILK BACTERIOLOGY: Pathogenic: Coxiella burnetti, Campylobacter, Brucella, Bacillus anthracis, Streptobacillus, Salmonella, Y. enterocolitica, Listeria, Vibrio, Mycobacterium bovis, and E.coli (EHEC). ___ – should be NEGATIVE, a positive result indicates improper pasteurization. Analysis of Milk quality: Milk is serially diluted and plated in culture media for colony counting. The satisfactory result should be comparable to __. Methods: __ (WBC count for infection), Agar plate count, Lab pasteurized count, __ (fecal coliform). Colony Counters: 1. Quebec Darkfield colony counter – ___ background, Manual (Wolffheugel grids) or Automatic counter 2. SC6 Colony counter – __ background, Pressure-sensitive counting, connected to computer/printer 3. __ – Manual counting of colonies in a plate. Traditional method like hemocytometer in Hematology. __ – dye is reduced (decolorized) by oxygen consuming bacteria; the speed of reduction is directly proportional to the number of bacteria present.

Phosphatase (ALP) test Grade A milk Somatic cell count; Coliform count dark Light or Dark Tally counter Methylene blue reduction test

Micriobiology Flashcards

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