Antimicrobial susceptibility of bacteria

How strongly treatments affect bacteria. Susceptible, Intermediate, Resistant

Disc diffusion method

Lawn innoculate an agar plate and place a disc containing antibiotic on it. let the disc sit for a day then measure the zone of inhibition. Compare the diameter (mm) to an interpretive chart

Zone of Inhibition

Full diameter (mm) of agar with no growth - does not include any area that look s hazy. Related to rate of diffusion.

Classification of organism as resistant to a particular antibiotic.

An antibiotic producing no plaque will most likely be ineffective against the bacteria causing your bunny's infection.

Classification of organism as intermediate to a particular antibiotic.

- there are some members of the bacterial population that are sensitive to this particular antibiotic, but others that are genetically immune to its effects.
- if the antibiotic used results in the death of sensitive members of the bacterial population then only the resistant ones will survive, resulting in the selection of a population resistant to that particular antibiotic.

Classification of organism as susceptible to a particular antibiotic.

The antibiotic has inhibited their growth and/or killed them, meaning that this particular antibiotic should be effective against the infection your rabbit has.

How to test disinfectants effectiveness against a given organism

Disk diffusion tests using MSA, MacConkey's Agar, Mueller Hinton Agar or a simple Nutrient agar with lawn innoculation.

Phenol Red broth: Interpretation and significance

Tests for the fermentation of different sugars (polymers of glucose or mannitol, etc).
If fermentation occurs acidic byproducts are created (pH decreases), notable by the bubble inside of the Durham tube.

Methol Red Test: Interpretation and significance

Tests for mixed acid fermentation.
Used to identify enteric bacteria based on their pattern of glucose metabolism. All enterics initially produce pyruvic acid from glucose metabolism.
Some use the mixed acid pathway to metabolize pyruvic acid to other acids (lactic, acetic, formic) <-- makes the solution more acidic which turns it RED/PINK, indicated a positive result.
Others use buytylene glycol pathway to metabolize pyruvic acid to neutral end-products <-- makes the solution more basic which turns it YELLOW, indicates a negative result.

Voges'-Proskauer Test: Interpretation and significance

Tests for the presence of acid byproducts (in particular ACETOIN) from enteric bacteria in the tube.
The test depends on the breakdown of glucose and the release of ACETOIN. If glucose is being broken down the addition of Reagent A & B will result in Red/Pink

Citrate Test: Interpretation and significance

Does the organism being tested utilize citrate for energy? If it does there will be a change in pH - alkaline byproducts will be released cause the agar to turn blue and potentially some growth

Urease Test: Interpretation and significance

If urease is produced it hydrolyzes the urea in the agar to ammonia which raises the pH of the medium and changes it from yellow to red (indicated a positive response).

Litmus milk test

Litmus milk is a complex medium that can potentially distinguish among many species of bacteria. Litmus milk has several components that can be metabolized: lactose (milk sugar); casein (milk protein); and litmus (a pH indicator that is purple to blue at neutral to alkaline pH and pink under acid conditions).

Reactions of the Litmus Milk Medium Test

Acid <-- there are acidic byproducts (pink)
Alkaline <-- starts to break down protein (blue)
Coagulation <-- solidification of medium (solid)
Reduction <-- becomes colorless (white)
Peptidigestion/Absorbtion <-- makes the medium clear

Litmus Milk Medium Test: Acid

lactose is fermented, leaving behind acidic byproducts and turning the medium pink

Litmus Milk Medium Test: Alkaline

lactose is not fermented and proteins are instead used for energy, the solution will become alkaline and more blue.

Litmus Milk Medium Test: Coagulation

Casein (milk protein) may be digested. This will coagulate the milk to form a curd (a solid).

Litmus Milk Medium Test: Reduction

The litmus (pH indicator) may be reduced and become colorless. The culture will then look milk white.

Litmus Milk Medium Test: Peptinization/Digestion

The casein (milk protein) may be metabolized all the way down to individual amino acids. This process, called peptonization, results in a clear (not milky) liquid that is usually brown in color.

Interpretation and significance: Nitrate Reduction Test

- The organism is able to reduces NO3 to N2 gas
- adding Reagent A & B turns the broth red (POSITIVE)
- IF adding Reagants causes no change in color, add ZINC, this will either cause the broth to turn red (reducing NO3 to NO2) because the organism couldn't cause the reduction on its own (NEGATIVE)
- or, if the NO3 had already broken down into some other nitrogenous compounds, there will be no change (POSITIVE)

Procedure: Nitrate Reduction Test

1. Inoculate nitrate broth with an isolate and incubate for 48 hours.
2. Add 10-15 drops each of sulfanilic acid and N,N-dimethyl-1-naphthylamine. If the bacterium produces nitrate reductase, the broth will turn a deep red within 5 minutes at this step.
3. If no color change is observed, then the result is inconclusive. Add a small amount of zinc to the broth. If the solution remains colorless, then both nitrate reductase and nitrite reductase are present. If the solution turns red, nitrate reductase is not present.

Interpretation and significance: Starch Hydrolysis Test

Lack of a zone of clearing when Iodine is added to an innoculated (and grown) agar plate means that the microbe was unable to hydrolyze starch and does not produce amalyse
If amalyse is present it will break the starch down into glucose molecules leaving a halo around the organism.

What comprises a SIM test?

Sulfur Reduction Test
Indole Production
Motility test

Interpretation and significance: Sulfur Reduction test (SIM)

If an organism can reduce sulfur to hydrogen sulfide, the hydrogen sulfide will combine with the iron to form ferric sulfide, which is a black precipitate. If there is any blackening of the medium, it indicates the reduction of sulfur and is a positive result.

Interpretation and significance: Indole Production test (SIM)

Drop Kovac's reagent on the top of the SIM agar. If the puddle turns stays yellow/clear NEGATIVE.
If the puddle turns pink POSITIVE

Interpretation and significance: Motility test (SIM)

- View the agar tube to determine motility of the organism. Negative result – the organism will not have deviated from the line of insertion. Positive result – anaerobic response: the organism diffuses through the agar; aerobic response: organism gathers on the top of the agar (forms a film).

Interpretation and significance: Catalase Test

Does the organism catalyze H2O2. A positive result will release O2 gas.
Pouring liquid onto the plate will result in small bubbles of O2.

Interpretation and significance: Oxidase Test

- Used to differentiate the bacteria that posses the cytochrome oxidase c enzyme from those who do not. If the organism is aerobic it will be able to reduce oxygen.
- The test detects the presence of the enzyme oxidase – a positive reaction will turn the colorless reagent purple.

***The enzyme cytochrome oxidase is involved with the reduction of oxygen at the end of the electron transport chain.