How to read api 20e results

A “Virtual” Unknown Bacteria Identification

Family Enterobacteriaceae

Phylum Gammaproteobacteria

Gram-negative

Enteric bacteria

Genera

Escherichia

Salmonella

Shigella

Yersinia

Edwardiella

Klebsiella

Proteus

Morganella

Providencia

Enterobacter

Hafnia

Citrobacter

In the following pages, you will see your own organism’s test results. Usually, a picture of the uninoculated medium will be alongside. You have to record whether your bacterium is + or – for the test result.

Be sure to read over the appropriate exercise in the lab manual first before interpretation of your test result. The exercise also gives you the purpose of the test, how it is inoculated, how it is read, what indicator or reagent is used, and so on.

Pages 15-17 are tables of biochemical tests for the major genera of the family of Enterobacteriaceae. Your unknown is one of those listed. You will use the tables to help you identify the bacterium.

The unknown report to turn in

To identify your unknown, use microscopic features (Gram reactions), cultural characteristics (colony morphology) and biochemical test results (positive and negative values).

Start with “Flow chart” for family of enterobacteriaceae (please see for chart at the end of this PPt). Plug in the positive and negative results/values in the Flow Chart to figure out the genus of your unknown.

To fully identify your unknown to genus and species levels, use the two tables/charts given at the endo of PPt. Plug in your results into tables correctly to find out your unknown. You may also use table in Bergy’s manual posted on ecampus under Unknown ID folder.

Finally, make sure you complete the identification sheet with all test results done for YOUR organism, including the genus and species name for it. Please see LAST page of PowerPoint (PPt). On a separate, make sure you tell about the biology and habitat of your identified organism in a few sentence (known bacterium)

Also, describe:

The kind of infection your uknown causes

How that infection is transmitted from person-to-person or from animals-to-persons

How those infections are prevented or controlled.

Unknown bacterium-code # 1

1. Gram staining

Assume you have virtually prepared a thin smear of your unknown; air-dried and heat fixed it.

Now Gram stain your smear (virtually). Make sure you add dyes and other reagents in the order they are applied and for the right time. Know the function/use of each dye and reagent added.

Consider image below is your unknown after staining and as seen under 100x magnification (oil immersion)

Now describe microscopic appearance/morphology of your unknown:

Gram reaction (positive or negative)

Shape of bacterial cells

Predominant and special arrangement of the cells

Refer to lab manual and/or chapter 4

(text book, pages 106-112)

2. Culture and isolation of your unknown on TSA plate

Virtually transfer your unknown aseptically onto TSA agar following the 4-step streaking method. Incubate overnight at 25/37 degree C. Below is an image of your unknown after an overnight incubation. Is the streaking done correctly? Why?

Now describe your unknown’s colony morphology visually or as seen under Quebec colony magnifying lens. Please use the following criteria.

Shape Texture

Margin Appearance

Elevation Pigmentation

Size Optical property

Elevation

Please refer to lab manual and/or chapter

(text book, pages 171-178)

3. Oxygen requirement

Virtually, inoculate one thioglycollate broth (include un-inoculated tube as a control) with your unknown.

In a similar manner, inoculate 3 TSA plates with your unknown; incubate at 25/37 degree C and @ 3 different oxygen environments: ambient (21% oxygen), gas pack (0% oxygen) and candle jar (5-10% oxygen). Below is an image of growth pattern of your unknown after overnight incubation.

Thioglycollate broth after

incubation

Now, which of the two broths show growth (turbidity)? Which of the 3 TSA plates show growth (colonies)?

What is thioglycollate and what does it do? By looking at the growth patterns of your unknown under the above 3 different conditions/environments, can you figure out which of your unknown is strict aerobe, strict anaerobe, or facultative anaerobe?

Please refer to lab manual and/or chapter (text book, pages 171-178)

Uninoculated thioglycollate

6

3. Biochemical tests

In these exercises/tests, you will use different tubed or plated culture media (with/without an indicator) to figure out the biochemical features (mainly color developments) of your unknown and use those features to identify the organism.

Based on your identification, you will write a lab report individually. Your report should include a list of all exercises/tests done, including microscopic, colony morphology, and biochemical features.

Here are a few questions to be answered while identifying

and preparing yourself for practical exam # 2.

What is the purpose of each test?

What is the name of medium used for that test?

What indicator is in the medium?

Is there a reagent to be added to confirm the test?

What does a + reaction look like?

What does a – reaction look like?

Biochemical test results and interpretations

Catalase test

On a clean slide, mix a loop-ful of your unknown with

a hydrogen peroxide (H2O2). Check for Bubbles formation.

What do those bubbles suggest/mean?

On a piece of blotting paper, add a drop

of oxidase reagent. Smear wet area with

your unknown. Check the kind of color

that develops within 20 seconds.

What does that color suggest/indicate?

Motility test:

Using a straight wire inoculate 3 TTC deeps (semi-solid agar) with your unknown

by stabbing from center top to down. Incubate overnight @25/37deg C and check

where bacteria are growing. Figure out if your unknown is non-motile or motile. Un-inoculated inoculated/Incubated

Refer to lab manual and/or chapter (text book, pages 171-178)

Un-inoculated After inoculation/incubation

Oxidase test

Motility test

Biochemical test results (Continued) - IMViC (Indole, methyl red, Voges Proscauer and citrate) tests

Citrate test

Inoculate the following media with your unknown and incubate overnight @25/37 deg C.

SIM (for indole production)

MR-VP (for Methyl red and Voges proscauer)

Citrate (for citrate utilization)

Indole test

Methyl red

Voges-Proskauer

Refer to lab manual for

results interpretation (as positive or negative)

Un-inoculated

After inoculation, incubation

Un-inoculated

After inoculation,

incubation

Un-inoculated

After inoculation,

incubation

After inoculation,

incubation

Un-inoculated

Biochemical test result results (continued)

Bacteria that produce an enzyme called nitrate reductase, reduce nitrate (NO3) to nitrite (NO2) and/or nitrogen (N2) gas. Production of enzyme is confirmed by adding nitrite reagents A and B. Red color development is positive and colorless is negative for NO2 production. If negative, zinc powder is added to confirm the presence of NO3. An empty space in the Durham tube indicates the production of N2 gas.

Nitrate reduction test

Urea hydrolysis test

Bactria that produce the urease, hydrolyze urea to ammonia (NH3). This is indicated by the development of hot pink color.

O-F media differentiate between oxidative bacteria (that produce acid from carbohydrates under aerobic condition only) and fermentative bacteria (that produces acid both under aerobic and anaerobic conditions).

Refer to lab manual for

results interpretation

Oxidative-Fermentative tests

Un-inoculated

After inoculation,

Incubation; reagent

added

10

Biochemical test results (continued)

Gelatin agar deep (hydrolysis/liquefaction)

Phenol red fermentation tests

Bacteria that produce gelatinase, hydrolyze/liquify gelatin in gelatin deep. Liquefied gelatin by gelatinase becomes runny. Gelatin can be liquefied at high temperature too. To tell liquefaction is because of gelatinase, test tubes are incubated on ice for about 10 min. If gelatin is still runny, it is because of gelatinase. Gelatin liquefied because of high temperature, re-gels or solidifies when incubated on ice.

Bacteria that utilize carbohydrates as a source of energy produce acid and gas (CO2). If acid is produced, broth becomes yellow (positive) and if gas is produced, it will accumulate in the top portion of the inverted Durham tube. If negative, broth will remain with the original re color.

Lactose broth

Refer to lab manual for results interpretation (+/-)

Biochemical test results (Continued)

Decarboxylase broths

Arginine

Lysine

Ornithine

Deaminase (phenylalanine) slant

Uninoculated

Uninoculated

After inoculation/incubation:

Lysine, Ornithine, Arginine appear like from left to right

After inoculation and incubation, with reagent (FeCl3) added

Please refer to lab manual for

results interpretation

Biochemical test results (continued)

Casein

Starch

Lipid

uninoculated

uninoculated

after incubation,

with added reagent

after incubation

after incubation

uninoculated

Refer to lab manual for results interpretation (as +/-)

ONPG (Beta galactosidase)

DNAse

Biochemical test results (continued)

Un-inoculated

Un-inoculated

After incubation

After incubation

Refer to lab manual for results interpretation (as +/-)

Family of Enterobacteriaceae

Bergey’s Manual of Bacterial Identification

Biochemical chart for unknown identification

(+) or (-) = usual reaction, d = different reactions

Oxidative-Fermentative tests

Un-inoculated

After inoculation/incubation