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Bacteriology and Mycology > Lab material > Flashcards

Lab material Flashcards

1
Q

What color are Micrococcus luteus colonies?

A

yellow

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2
Q

What are hemolysins?

A
  • extracellular enzymes produced by bacteria that are capable of lysing RBC
  • used in the classification of streptococci
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3
Q

What is the Beta hemolytic group?

A
  • the bacteria that form a soluble hemolytic substance capable of completely lysing RBC
  • Colonies will be surrounded by a zone of complete hemolysis
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4
Q

What is the Alpha hemolytic group?

A
  • bacteria that produce a green discoloration of the agar in the immediate vicinity of the colongy
  • The greening is followed by a partial lysis of the cells in the area
  • Lysis is supposedly due to hemolyzing concentrations of acid
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5
Q

What is a general pupose medium?

A
  • The exact chemical composition is unknown, and can vary from batch to batch.
  • Most bacteria can grow on it
  • Ex:
    • Blood agar
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6
Q

What is a differential medium?

A
  • allows differentiation of bacterial species or types based on phenotypic characteristics
  • use pH indicators to siignify whether bacteria degrade nigtrogenous compounds or charbohydrates
  • Indicators:
    • Phenol Red
    • Bromothymol Blue
    • Bromocresol purple
    • Methy Red
    • Neutral Red
    • Litmus
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7
Q

What is a selective media?

A
  • Used for the isolatin of specifc types of bactria
  • contains a chemical that inhibits growth of a specific group of bacteria
  • May also be a differential media
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8
Q

What is the CAMP test?

A
  • Christie, Atkins, andMunch-Peterson
  • Used to identify group B streptococci
  • S. aureus produces B-toxin whose hemolytic activity is enhanced by an exracellular factor produced by group B strptococci
    • Test is made by making a single streak of S. aureus down a blood agar plate, and then placing a single streak of the group B Streptococcus suspect perpedicular to it. Must NOT touch, but be very close. Then incubate at 37C
    • Positive if complete hemolysis develops
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9
Q

What is the Carbohydrate Fermentation Test?

A
  • Conducted with Phenol Red Broth
  • Test if a particular organism will or will not produce acid from the particulr carbohydrate present in the medium
    • variety of medium compositions
    • Add: Glucose, Maltose, Mannitol, Trehalose (more than 50 different ones)
  • Inoculate with a small amount of bacteria from a medium tht does not contain other fermentable carbhydrates (Ex: MAC)
    • Incubate 37C 24h
    • Red/Pink = Negative
    • Yellow = Acid production = Positive
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10
Q

What is the Catalase Test?*

A
  • identifies bacteria that produce the enzyme catalase and are able to degrade hydrogen peroxide
    • H2O2 produced by aeroves, facultative anaerobes, and microaerophiles during oxidative metabolism of carbohydrates
    • Highly toxic to the cell
  • Differentiates between the following genera:
    • Staphylococcus (+) from Streptococus (-)
    • Bacillus (+) and Clostridium (-)
    • Listeria / Corynebacterium (+) from Erysipelothrix (-)
    • Most G- are Catalase +
  • Place Hydrogen Peroxide on a slide and add inoculum. Observe for bubbles
    • Immediate bubbles = Positive
    • No bubbling = Negative

Blood Agar colonies can cause a false positive test, due to RBCs containing catalase

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11
Q

What is Chocolate Agar?

A
  • Contains the same ingredients as blood agar
    • RBC are added at 85C to partiallly lyse them and release hemin (x factor) and NAD (V factor) which are required for some gastidious bacteria
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12
Q

What are Citrate Agar Slants?

A
  • Used to deterime if an organism is capable of utilizing citrate as the sole source of carbon for metabolism with a resulting alkalinity
  • Inoculate slant by streaking, icubate 37C for 24-48h
    • Changes to Blue = Acetate production = Positve
    • Stays Green = Negative
  • Selective and differential
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13
Q

What is the Gram Stain?

A
  • Categoizes bacteria into 2 broad groups:
    • Gram + (blue-purple)
    • Gram - (pink -red)
  • Gram stains work by creating a large I2-Crystal Violet complex inside the cell
    • The alcohol is dissolves lipids in the outer membrane of Gram - baceria, allowing the I2-Crystal Violet complex to leak out of the cell.
      • Gram + have thick proteoglycan and polysaccharide membranes, no lipid = no leakage
    • The safranin is then added and colors only the the G- cell that were decolorized pink
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14
Q

What is Gram Variable?

A
  • Gram + organism staining Gram -
    • Treatments can disrupt the cell wall of Gram+ organisms (Ex: lysozyme, autolytic enzymes, growth conditions, age) and allow leakage of teh I2-Crystal Violet complexes causeing Gram + organisms to stain Gram -
    • Gram + cells may appear Gram - after antibiotic therapy
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15
Q

How do you perform a Gram Stain?

A
  1. Place inoculum on slide
    • Broth samples just need a loopful of medium
    • Plate samples need to be added to a drop of water and mixed thoroughly
  2. Air dry at room temp
    • if liquid is present during heat-fixing the cells will distort or lift off the slide
  3. Heat fix
    • pass slide through the flame to adhere the cells to the slide
    • Warm, NOT hot - will distort normal shape and structure
  4. Gram stain:
    1. Flood slide with Crystal Violet for 1 minute
    2. Drain and rinse with tap water
    3. Flood slide wwith Iodine for 1-2 minutes
    4. Rinse with tap water
    5. Decolorize with 95% ethyl alcohol (use dropper) until stain is no longer bein eluted from the smear
    6. Rinse with tap water
    7. Flod slide with safranin for 1 minute
    8. Rinse with tap water, air dry (or blot, but do not rub)
  5. Examin under oil-immersion
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16
Q

What is the Indole test?

A
  • Determines if a given species of baceria possesses the enzyme tryptophanase
    • Will deaminate the AA tryptophane to produce indole and possibly skatole
  • Medium must contain tryptophan - (Ex Tryptone Broth 1%)
  • Inoculate the tube with a generoush sample.
    • Incubate 37C for 24-48 h
    • Add 5 drops of Kovac’ reagent
      • Red ring = Positive
      • No red = Negative
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17
Q

Why does Serratia marcescens cause a false positive Indole test?

A

It’s red pigment extracts into the Kovacs’ solution and give it a reddish appearnce and constitutes a false positive

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18
Q

What is MacConkey Agar

A
  • Growth medium of bile salts and crystal violet
    • inhibit most Gram positive and some gram negative bacteria
  • Neutral red is pH indicator
    • Lactose fermentors will become pink
    • Can lose color due to changes in the A/B balance
  • Differentia and Selective
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19
Q

What is Mannitorl Salt Agar?

A
  • Selective medium to isolate coagulase-positive staphylococci
  • Contains 7.5% NaCl - inhibits nearly all other groups of bacteria and some speceies of staphyloccci
  • Contains mannitol and organisms which produce acid fro manitol will produce an acid change in the medium
  • Inoculate a batriera spot about the size of a dime. Incubate 18-24 h at 37C
  • Differential and Selective
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20
Q

What is the Motility Test?

A
  • Purpose is to determine if an organism is motile or non-motile under the growth conditions of the test
  • Contains 0.5% agar and is semisolid - allow movement
  • Inoculate with a needle by stabbing directly down the center of the tube down to as close to the bottom as possible, and draw the needle out in the same direction
    • incubate at room temperature for 24-48h
    • Turbidity from the stab = Positive
    • Turbid only in stab = Negative
  • General purpose differential

Due to lack of oxygen at the bottom of tube, organisms that are stricktly aerobic and motile may appear non-motile (false negative)

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21
Q

What is an Oxidase test?

A
  • Identifies bacteria which produce the enzyme cytochrome oxidase and are able to oxidize a dye tetramethyl p-phenylenediamine to indophenol (colored)
  • Differentiate Enterobacteriaceae from:
    • Neisseria spp. and Morazella catarrhalis are oxidase positive
    • Psuedomonas spp(except Burkholeria cepacia)
    • Aeromonas
  • Use a cotton swab to collect a visible amount of growth from Blood agar plate
    • Add 2 drops oxidase
    • Purple (within 15 seconds) = Positive
    • Purple (1-2 minutes) = False positive (except Pasteurella/Mannheimia spp)
    • No change = Negative

Dyes in MacConkey can lead to false negative due to excessive acid

Tetramethyl p-phenylenediamine is unstable - will autoxidize - do not use if deep blue

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22
Q

What is Phenol Red Broth with Mannitol?

A
  • Distinguish between Bacillus anthracis or Bacillus cereus (negative) from Bacillus subtilis (positive)
  • Contains pancreatic diget of casein, sodium chloride, a carbohydrate, and phenol red
  • Contain Durham tubes to visualize gas production
  • Innoculate with isolated colony from a young culture
    • Inncubate 35-37C for 3-5 days
  • Yellow = Positive
  • Red = Negative
  • Gas = gas fermentation reations
  • General purpose differential
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23
Q

What is Phenol Red Broth with Trehalose?

A
  • Ability to ferment Trehalose to produce acide can distinguish between Streptococcus suis (positive) and Streptococcu zooepidemicus (negative)
  • Has a sugar-free basal medium, phenol red, and 1% trehalose
  • Inoculate with an isolate from a yong colony and incubate at 35-37C for 3-5 days
    • Yellow = Positive
    • Red = Negative
  • General purpose differential
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24
Q

What is a Spore Stain?

A
  • 2 major spore forming genera Bacillus and Clostridium - cause botulism, gangrene, tetanus, anthrax, etc
  • Malachite green is weak binding to cell/spore wall
    • Use heat to help lock in the stain to the spore wall
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25
Q

How do you perform a Spore Stain?

A
  1. Prepare smear, air dry, and heat fix by passing through flame 3x
  2. Cover smear with Malachite green and pass 20x 4 inches above flame
    • should steam for ~30 seconds but not dry out
  3. Let cool
  4. Rinse with tap water
  5. Flood slide with safranin for 30 seconds
  6. Rinse, Dry, examine under oil immersion
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26
Q

What are Triple Sugar Iron (TSI) Agar Slants?*

A
  • Indicates the ability of an organism to ferment lactose, sucrose, and dextrose with formation of acid, possible gas, and also the ability to produce hydrogen sulfide
  • Inoculate slant with loopful of culture
    • inoculate the butt by stabbing with needle
    • Incubate at 37c for 24h (may take longer)
  • Reactions:
    • No change = No change in color
    • Alkaline (K) = more Pink/Red
    • Acid (A) = Yellow
    • Hydrogen Sulfide (H2S = Black
    • Gas (G) = gas bubbles in butt
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27
Q

What are the interpretations of TSI slants?*

A
  • NC/NC: either does not act on any of the carbs, or is an O-F oxidizer and requires oxygen
  • K/NC: Organism has acted on the proteinaceous compounds. O-F should be run as above
  • K/A: Organism is a Fermenter and produces acid from only glucose not from lactose or sucrose. Also producing alkaline products
  • K/A + H2S: Organism is a Fermenter and produces acid from only glucose not from lactose or sucrose. Also producing alkaline products. H2S is produced from sodium tiosulfate. If it obscures results run an O-F
  • K/NC + H2S: Organism has acted on the proteinaceous compounds. O-F should be run. H2S is produced from sodium tiosulfate. If it obscures results run an O-F
  • A/A: Bacteria are known to be fermenters in O-F medium, Ferment lactose and/or sucrose and glucose
  • A/A + H2S : Bacteria are known to be fermenters in O-F medium, Ferment lactose and/or sucrose and glucose and produce H2S.
    • Erysipelothorix rhusiopathiae small black line
    • Proteus vulgaris large amount
  • A/A + G: Bacteria are known to be fermenters in O-F medium, Ferment lactose and/or sucrose and glucose ad produces gas
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28
Q

What are Urea Agar Slants?

A
  • Deterimes if an organism has the ability to produce the enzyme urease which will split urea into two molecules of ammonia
  • Inoculate slant heavily with growth from any medium Incubate 37C for 24-48h
    • Pink = ammonia = Positive
    • Yellow = Negatie
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29
Q

What is Rappaport-Vassiliadis (RV) Enrichment Broth?

A
  • Used for the isolation and cultivation of Salmonella species from food, clinical specimens, and environmental samples
  • Enrichment results in a higher tolerance to malachite green by Salmonella relative to other members of Enterobaceriaceae
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30
Q

What is a complex medium?

AKA General Purpose medium

A
  • Exact chemical composition is not known
  • Contains nutrients such as digest or extracts from animal or plant products which provides sugars, proteins, and vitamins
  • Most bacteria can grow on it
31
Q

What is a differential medium?

A
  • Allows differentiation to bacterial species or types based on phenotypic characteristics
  • End-products of nitrogen compounds tend to be alkaline in nature
  • The end-products of carbohydrate degradation tend to be acid in nature
32
Q

What is a selective medium

A
  • Used to aid in isolation of specific types of bacteria
  • Contain chemicals that inhibit the growth of a specific group of bacteria
33
Q

What are some gram positive anaerobes?

A
  • Rod shaped
    • Clostridium. spp
      • spore forming
    • Actinobaculum suis
      • non-spore forming
  • Cocci:
    • Peptostreptococcus asaccharolyticus
    • P. indolicus
34
Q

What are some gram negative anaerobes?

A
  • Rods, non-spore forming:
    • Bacteroides fragilis
    • Dichelobacter nodosu
    • Fusobacerium necrophorum
    • F. equinum
    • Porophyromonas levi
    • P. gulae
    • Prevotella melaninogenicus
  • Spirals:
    • Brachyspira hyointestinalis
    • B. piloscioli
35
Q

What are some clinical conditions that are suggestive of Anaerobic Infections?

A
  • Proximity to mucosal surfaces
  • Putrid odor
  • Presence of gas
  • Failure to get growth by aerobic culture
36
Q

What needs to be collected for Clostridial infections like Blackleg, Malignant Edema?

A
  • Affected tissue or aspirate from affected tissue
    • Muscles or SQ tissues
37
Q

What samples should be collected for Necrotic enteritis / Enterotoxemia?

A
  • place several ounces of fresh small-intestinal contents in a jar or plastic bag, or tie off a section of the affected intestine
  • Submit ASAP on ice
38
Q

What specific infections are anaerobes involved in?

A
  • Clostridium spp
  • Dichelobacter nodosus - Foot rot in sheep
  • Fusobacterium necophorum - Calf diphtheria, liver abscesses and foot rot in cattle
  • Actinomyces suis - Urinary tract infections in sows
39
Q

What non specific infections involve anaerobes?

A
  • Caused by nonspore forming, gram negative rods and cocci and are often polymicrobial infections
40
Q

What is Transport medium for anaerobes?

A
  • Tube with Needleport sepum hungate cap
  • anaerobic environment in the tube
41
Q

What are anaerobic techniques

A
  • Protect samples from air
  • Plates are incubated under oxygen-free atmosphere
42
Q

What is the anaerobic jar technique?

A
  • place petri dishes inside
  • add Gas Pak envelope, Methylene blue strips and Catalyst holder with Palladium
  • Screw on lid
  • Advantages:
    • Simple
    • Low Cost
  • Limitations:
    • Good for aerotolerant anaerobes
    • Strict anaerobes will not grow
    • Difficult to check plates during incubation
43
Q

What does Palladium catalyst do?

A
  • Hydrogen released from Gas Pak interacts with Palladium catalyst
  • O2 + 2H2 ⇢ 2 H2O2
  • This depletes the O2 to create an anaerobic condition in the jar
44
Q

What is the point of the methylene blue strip in an anaerobic jar?

A
  • Indicates Anaerobiosis
  • Blue to white change indicates anaerobic condition was achieved
  • Can also be done with Resazurin
    • Pink to white change
45
Q

Why does Fusobacterium necrophorum grow under Anaerobic conditions but not aerobic conditions?

A
  • Oxygen is toxic to F. necrophorum
    • Produces hydrogen peroxide and Superoxide ions (toxiic substances)
    • anaerobic bacteria lack catalase and superoxide dismutase that would detoxify conditions
46
Q

Why do anaerobic infections have putrid odors?

A
  • Fermentation products
  • Acids: Short chain fatty acids (i.e. butyric acid) produced from carbs
  • Gases
  • Amines (Putrescine, Cadeverine, etc)
47
Q

What diseases does M. heamolytica cause in cattle?

A
  • Bovine Respiratory Disease
  • Shipping Fever
  • Mannheimiosis
48
Q

What diseases does P. multoida cause in rabbits?

A

Snuffles (upper Respiratory infection)

49
Q

What are the samples to collect from animals that have respiratory problems?

A
  • Alive:
    • Nasal discharge or nasal swabs
    • Tracheal swabs
    • Tracheal washings
  • Dead:
    • Tracheal swabs
    • swabs of lung lesions
    • Lung pieces
50
Q

What samples should be collected when an animal aborts?

A
  • Fetus (Whole ideal)
    • Need: Abomasal contents or heart blood
    • Lung, liver, etc, whole placenta or sections that show lesions
  • Placenta
  • Uterine/vaginal discharge
51
Q

What is a Buffered Brucella Antigen (BBA) card test?

A
  • Agglutination test (Positive test agglutinates)
  • Simple and rapid method
  • Use for detection of antibodies in blood
  • Qualitative
52
Q

What is the Campy PakR Jar Technique?

A
  • Campylobacter is microaerophilic
  • The gas pack used produces an O2 atmosphere between 5 to 15%
53
Q

Why would some samples from an abortion need to be incubated at an increased CO2 atmosphere?

A
  • Brucella sp. are carboxyphilic - Need increased CO2 to grow
54
Q

How do you assess the clinical relevance of isolations of E. coli, K. penumoniae, P. aeruginosa which can be causative agents or contamination?

A
  • Depends on sample collection
    • Samples collected aseptically- not likely contaminants
    • Placenta picked up from the ground - likely contaminants
  • Usual suspects not obtained (Brucella Campylobacter, etc)
  • Isolation in pure or almost pure culture
55
Q

If joint fluid is cultured and no bacteria are present, does that eliminate bacterial infection in the arthritic condition?

A
  • No
  • Antigen and antibody complex will induce proinflammatory cytokines (Type III hypersensitivity)
56
Q

What are the problems with PCR assay?

What can be done to avoid them?

A
  • PCR product contamination can result in false positives
  • Instituting proper control procedures
57
Q

What are the advantages of PCR procedure over other methods of detecting bacteria?

A
  • PCR is rapid and is the most sensitive
  • PCR positive means the organism is present in the sample
58
Q

What is Agarose Gel Electrophoresis?

A
  • Method to separate mixtures of charged molecules according to molecular size
    • Protein, RNA, DNA
  • Molecules move through a gel (permeable matrix) when an electric current is passed across it
  • Molecules travel through pores in the gel at a speed that is inversely related to their lengths
59
Q

What is the agarose gel made of?

A
  • Agarose
    • Porous to allow DNA migration
    • The higher the concentration the denser the matrix
  • Buffer
    • Provides ions in solution to ensure electrical conductivity
  • Stain to visualize DNA on gel
    • Ethidium bromide
      • Intercalates into DNA
      • Fluoresces under UV light
60
Q

What is the loading dye for Agarose Gel Electrophoresis?

A
  • Bromophenol blue
61
Q

What is a DNA ladder marker?

A
  • DNA of known sizes
  • Determine the size of unknown DNA
62
Q

What are the disadvantages of PCR?

A
  • Equipment
  • Cross-reactions
  • Contamination - False positive
  • Taq polymerase - Expensive
  • Non-specific amplications
63
Q

What are the Advantages of PCR?

A
  • Quick
  • Reliable
  • Sensitive
  • Relatively easy
  • Specific
64
Q

What are the 3 steps of PCR?

A
  • Denaturation
    • 95C
    • Double-stranded DNA is separated into 2 single strands
      • Break hydrogen bonds between the nucleotide bases pairs
  • Annealing
    • 40 - 60C
    • Enables DNA primers to attach to the template DNA
      • primers anneal (bind) to their respective complementary sequences in the single strands of DNA
  • Extension
    • 72C
    • DNA polymerase extends (elongates) the DNA chain by adding nucleotides
65
Q

What are the 3 steps of PCR?

A
  • Denaturation
    • 95C
    • Double-stranded DNA is separated into 2 single strands
      • Break hydrogen bonds between the nucleotide bases pairs
  • Annealing
    • 40 - 60C
    • Enables DNA primers to attach to the template DNA
      • primers anneal (bind) to their respective complementary sequences in the single strands of DNA
  • Extension
    • 72C
    • DNA polymerase extends (elongates) the DNA chain by adding nucleotides
66
Q

What is Polymerase Chain Reaction?

A
  • In vitro method to amplify a specific DNA sequence
  • Components:
    • DNA template
    • Primers
    • DNA polymerase enzyme (Taq polymerase)
    • Nucleotide bases
    • Reaction buffer
67
Q

What is Acid Fast Staining?

A
  • Used for Mycobacterium species
    • Do not stain well with Gram’s stain
    • Cell wall of Mycobacterium contains a waxy lipid layer, called mycolic acids, located above the peptidoglycan layer
    • Need a stronger decolorizing agent. Acid-alcohol is used
  • Flood smear with Kinyoun’s carbol-fuchsin and let stand for 4 minutes
  • Rinse gently with water
  • Decolorize with Acid Fast Decolorizer, drop by drop until alcohol runs colorless from the slide
  • Rinse with water
  • Counterstain with methylene blue for 20-30 seconds
  • Rinse with water
  • Air dry and examine
68
Q

How does acid-fast differ from gram staining?

A
  • Both are differential staining
  • Gram staining: Gram positive and negative
  • Acid-fast staining: Acid-fast and non-acid-fast
  • Acid-fast bacteria contain waxy lipid layer called mycolic acid
69
Q

Is acid-fast staining of diagnostic value?

A
  • yes
  • only 2 genera are acid-fast:
    • Mycobacterium
    • Nocardia
70
Q

What are the causative agent(s) of canine ehrlichiosis?

A
  • Ehrlichia canis
  • Ehrlichia ewingii
  • Ehrlichia chaffeensis
71
Q

How does the age of cattle influence when they are vaccinated against Anaplasmosis/Ehrlichiosis

A
  • Vaccination with live organisms to younger calves provide immunity against clinical disease.
  • A herd may be protected using this approach in countries where where the pathogen is endemic
72
Q

What is a possible source for A. marginale?

A
  • Dermacentor andersoni tick
  • Mechanical transmission
73
Q

What is a possible source for E. chaffeenis?

A

Amblyomma americanum tick

Bacteriology and Mycology (35 decks)

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