Lecture 2 - Identification, Disinfection Flashcards by Christine Parascandola

What are the two basic types of bacteriological culture medium?

Defined/simple (e.g. minimal salts).
Enriched/complex (e.g. nutrient broth).

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What is an example of differential media?

MacConkey agar, Blood agar

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What is an example of selective media?

Selenite broth, MacConkey Agar

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What is an example of enriched media?

Blood agar

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What is an example of transport media?

Stuart’s media

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Describe streaking

Go in a clockwise rotation. (quadrant 1-4).
E.g. Ecoli 1+ (only in 1 quadrant), Ecoli 4+ (in all four quadrants).

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Will gram positive bacteria grown in MacConkey Agar?

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How can the surface of a colony be described?

Surface: smooth & glistening; rough;
granular; wrinkled dry & powdery

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How can an entire colony be described?

Whole colony: circular, filamentous;
irregular; rhizoid; punctiform

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How can the elevation of a colony be described?

Elevation: flat; raised; convex;
pulvinate, umbonate

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How can the margins of a colony be described?

Margin: entire, undulate, curled,
lobate, serrate, filamentous

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How can the size of a colony be described?

Size: diameter in mm; pinpoint;
small, medium and large

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How can the color of a colony be described?

Color: pigmentation;
e.g. Staphylcoccus aureus

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Decribe the two types of Motility Tests

Wet mount: direct
microscopic observation . to see if it’s motile
.
.. … . .. .
Neg. Pos.
.
.
Nonmotile Motile
Motility culture medium:
soft agar stab –> if motile it will spread out

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Describe the different types of Serological Tests used for Bacterial Identification. Explain the rationale behind these tests

Rationale: each species has unique antigenic components , eg.
H- antigens = flagella = protein
O- antigens = LPS = polysaccharides
K- antigens = capsule = polysacch. or protein
• Types of tests:
- Slide agglutination: simple, inexpensive
Used for initial screening of colonies that may be Salmonella
ELISA, counterimmunoelectrophoresis: complex, expensive

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Name some common serological assays

ELISA, latex agglutination, Western Blotting,
Older assays not commonly used any more

Don’t need to isolate pathogen - can use patient’s serum!
Counter-immunoelectrophoresis, radioimmunoassay,
complement fixation, fluorescent antibody, etc.
ELISA, latex agglutination, Western Blotting,
Older assays not commonly used any more

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Detecting Carbohydrate Utilization

Medium with a ph indicator
if ph drops, turns medium yellow

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How can you differentiate between fermentors and non-fermentors?

lactose and sucrose are 10 x the concentration of glucose so it is easy to differentiate fermenters from non-fermenters

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Reactions Produced on TSI Medium
Non-fermenters = entire medium stays?

red

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Reactions Produced on TSI Medium
Non lactose fermentors - stain is what color?

if only ferments glucose, not lactose or sucrose, only butt turns yellow b/c not enough acid to turn aerobic area yellow.

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Reactions Produced on TSI Medium
Lactose/sucrose fermentors - medium is what color?

so muhc acid produced turns eveyrthng yellow.

if too much acid turns butt black

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Bromthymol Blue is useful for ?

pH indicator in Oxidative phosphorylation medium
ferments = yellow
utilizes amino acids = turns blue
nothing = stays green

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Physical Agents
• Temperature: moist, dry, incineration
• Radiation: ultraviolet, gamma, X-rays
• Mechanical: sonication, filtration

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What is the purpose of Decontamination?

Cleaning and any additional steps required to
eliminate risk of infection while handling
devices or attire. A reduction in potentially
pathogenic organisms to a level that is safe to
handle.

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What is the purpose of Disinfection?

Elimination of most if not all PATHOGENIC organisms, including spores. Most effective when preceded by cleaning.

What is the purpose of Sterilization

Elimination of ALL living organisms

What is the purpose of incineration?

• Incineration - > 300o C until completely oxidized - fast, but expensive - useful in elimination of pathogen contaminated materials e.g. bandages, carcasses, tissues

What is the purpose of Ionizing Radiation (Ozone)?

Interact with macromolecules and cause disruption of covalent bonds! • Useful in sterilization of inanimate, heat sensitive materials e.g. syringes, catheters O2- . oxygen radical X-rays, gamma-rays OH. hydroxyl radical Short lived radicals

What is the purpose of Liquid Filtration?

Choose filter with avg. pore size of 0.2 microns. • Exclude most bacteria - avg. smallest dimension is 0.5 microns. • Applications: sterilize liquids which are heat sensitive.

What is the purpose of Air Filtration

HEPA = high efficiency particulate air filter = 99.999% of 0.12 micron particles

What Chemical Agents can be used?

Alkylating agents - gaseous and liquids • formaldehyde (formalin), glutaraldehyde, ethylene oxide • extremely reactive with carboxyl, hydroxyl and sulfhydryl groups of proteins ----> alkylate = addition of hydroxyl, methyl, or ethyl groups or cross-linking of reactive groups • sterilizing research or hospital equipment that is sensitive to heat: plastic syringes, equipment O Protein X Protein Y

What does MEC stand for?

MEC-minimum effective concentration of a germicide required to achieve advertised microbicidal activity.

A Low-level disinfectant is a germicide that kills most ________ bacteria and ____-_______ and ______ sized viruses.

Germicide that kills most vegetative bacteria and lipid-enveloped and medium size viruses.

What is an Intermediate level disinfectant?

It kills all microbial pathogens except spores

What is a High level disinfectant?

kills all microbial pathogens except large number of bacterial spores

In general, disinfectants have a gradient of microbicidal activity on bacteria of different cell types:

Gram Positive < Gram Negative < Acid Fast < Spore Formers

Alcohols, such as ethanol, isopropanol, denature?

proteins and solubilize lipids

Alkalies - sodium hydroxide, 1-2%; “lyse” • destroys ? • used on ?

cell walls and cell membranes, inanimate objects not sensitive to alkaline pH

Heavy metals - HgCl2 (0.001%), AgNO3, and copper • metals function to poison ______ activities by interacting with ______ groups of ______ residues. • the basis for merthiolate and mercurochrome used as skin antiseptics?

enzyme, sulfhydryl, cysteine “burns” open tissue as it is solubilized in alcohol !!!

Oxidizing agents • Halogens - iodine and chlorine inactivate enzymes by converting functional ____ groups to oxidized ____ forms; can attack ____ groups, ______ groups and ______ residues;

-SH, S-S, -NH, indole, tyrosine

Surface active agents - induce a reduction of ______ or _______ _____ e.g. _______ agents, _______.

surface, interfacial tension, wetting, detergents

Anionic agents are _____ or ____ acids with a ______ charge

soaps, fatty, negative

Phenolic compounds, such as ______, _____, and _______, cause membrane ________ and protein _______. Usually mixed with _____ to increase penetration. Not usually inactivated by ______ ______. No longer used because of its ______. Lysol is _____. ______ is incorporated into soaps, antiperspirants, toothpastes, filters.

phenol, creosol, hexachlorophene, disruption, denaturation, soap, organic matter, toxicity, creosol; hexachlorophene

Aldehydes: the most commonly used are ?

glutaraldehyde, ortho-Phthalaldehyde, and formaldehyde FOG

Glutaraldehyde is a high level _______ that is compatible with many materials, and so is the agent of choice for ______ disinfection for ______ materials.

disinfectant, chemical, hospital

ortho-Phthalaldehyde is a ____% solution for ___ min is a high level disinfectant, is also compatible with many materials, is more active that ____ against mycobacteria.

0.55, 12, GAH

Formaldehyde

high level disinfectant used as a liquid or gas. Not used as commonly as previously due to irritation and potential carcinogenicity.

Why is mycobacteria so resistant?

• Mycobacteria -> glycolic acids on the surface make it hearty so it is much more resistant .

How does moist heat help to kill bacteria?

Moist heat denatures proteins and if proteins dont work bacteria dies

What is an autoclave good for? What cannot be autoclaved?

• Good for metal objects • Contents under pressure • No plastic

Which is quicker at disinfection/ sterilization, dry or moist heat? what is the difference in time?

Dry Heat 1-2 hours slower than moist heat.

What are the limitations to sterilization with dry heat?

Limited to inanimate objects/ heat resistant objects

What is incineation? What is the benefits/ cons?

• Incineration: Until completely oxidized ( > 300 degrees celcius) ◦ Fast but expensive ◦ useful to eliminate pathogen contaminants/ ect. useful in elimination of pathogen contaminated materials e.g. bandages, carcasses, tissues

What area in the radiant energy spectrum has the best bacterial effect? What is the practical methods of sterilization? Impractical?

• < 300 nm large bacterial effect • UV is practical • xray/ gamma will work but not practical > 300 nm - little bacteriocidal effect < 300 nm - large bacteriocidal effect

How does ionizing radiation ( ozone) work to kill bacteria? What can it be used for? Is it common?

nteract with macromolecules and cause disruption of covalent bonds! • Useful in sterilization of inanimate, heat sensitive materials e.g. syringes, catheters. Again, not practical so not used often

What is sonification? Is sonification common?

Bubbles bombard cells and lyse them. No it is not common

What are the physical means of disinfection?

Physical means include temperature, radiation, filtration & sonication.

Why does an autoclave sterilize efficiently?

• An autoclave sterilizes efficiently because of efficient transfer of latent heat from water to object.

What is the most effective way to clean?

Remove organic material first before using antiseptic. Antiseptic wont be very effective if there is organic material present. The organic material will be protective.

What % alcohol is effective against strep. pyogenes?

100% is not very effective against streptococcus pyogenes. ◦ Lower concentrations are until about 50 % ◦ 60% and 95 % is the optimal concentrations