I need this unknown ID assignment completed, please
rissak54
UnknownID03.pptxA “Virtual” Bacterial Unknown 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.
3
Unknown ID # 3
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
Please refer to lab manual and/or chapter 4
(text book, pages 106-112)
Cultures 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
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.
uninoculated
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?
Refer to lab manual and/or chapter (text book, pages 171-178)
3. Biochemical tests
In these exercises/tests, you will use different tubed and/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?
Oxidase test
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. Motility test
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.
Refer to lab manual and/or chapter (text book, pages 171-178)
uninoculated
after incubation
Biochemical test results (continued) – Indole, methyl re, voges proscauer and citrate (IMViC)
Indole test (SIM) MR-VP tests
Methyl red test Citrate test
Un-inoculated after incubation,
with reagent
uninoculated
uninoculated
uninoculated
after incubation,
with reagent
after incubation
after incubation,
with reagent
Biochemical test results (continued)
Nitrate test (NO3 reduction)
Urea hydrolysis test
uninoculated
uninoculated
uninoculated
after incubation
after incubation
after incubation,
with reagents
O-F glucose at right (oxidation or fermentation)
Refer to lab manual and/or chapter (text book, pages 171-178)
Biochemical test results (continued)
Carbohydrate broths
Glucose
Lactose
Sucrose
Mannitol
Gelatin agar deep (hydrolysis)
uninoculated
uninoculated
after incubation
after incubation
Refer to lab manual and/or chapter (text book, pages 171-178)
Biochemical test results (continued)
Decarboxylase broths
Arginine (A)
Ornithine (O)
Lysine (L)
Deaminase (phenylalanine)
uninoculated
uninoculated
after incubation,
all 3 amino (AOL) acid broths look the same
after incubation,
with reagent
Refer to lab manual and/or chapter (text book, pages 171-178)
Biochemical test results (Continued)
Casein
Starch
uninoculated
uninoculated
after incubation,
with reagent
after incubation
uninoculated
after incubation,
with reagent
Lipid
ONPG (Beta galactosidase)
DNAse
Biochemical test results (continued)
uninoculated
uninoculated
after incubation
after incubation
Refer to lab manual and/or chapter (text book, pages 171-178)
Family of Enterobacteriaceae
Bergey’s Manual of Bacterial Identification
Biochemical chart for unknown identification
(+) or (-) = usual reaction, d = different reactions
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?
