I need this unknown ID assignment completed, please
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UnknownID02.pptxA “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 # 2
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
Refefer 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 atmospheric conditions/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
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 conditions/environments, can you figure out whether your unknown is strict aerobe, strict anaerobe, or facultative anaerobe?
Refer to lab manual and/or chapter (text book, pages 171-178)
Your organism on 3 different TSA plates in 3 different environments
uninoculated
after incubation
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, cultural (colony morphology) and biochemical features.
Here are a few questions to be answered while identifying
and preparing 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 (continued)
Catalase test Oxidase test
On a clean slide, mix a loop-ful of your unknown with a hydrogen
peroxide (H2O2). Bubbles formation is positive for an enzyme called catalase. On a piece of blotting paper add a drop of oxidase reagent. Smear with your unknown. Blue color formation within 20 seconds is an indication of oxidase production by your unknown.
Motility test
Using a straight wire, inoculate 3 TTC deeps (semi-solid media) with your unknown
by stabbing from center top to down. Incubate overnight @25/37deg C and check
where bacteria are growing. If unknown grows following stab-line, it is non-motile.
(negative). If growing in the entire medium, it is motile (positive). by stabbing from
center top to down. Incubate overnight @25/37deg C and check
Refer to lab manual for
results interpretation (as positive or negative)
Un-inoculated After inoculation/incubation
8
Biochemical test results (continued) – Indole, methyl red, voges proscauer and citrate (IMViC) tests 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 (SIM) Methyl red test
uninoculated
uninoculated
uninoculated
after incubation
after
incubation, reagent added
after incubation,
reagent added
after incubation, reagent added
Citrate test
Voges-Proskauer/VP test
uninoculated
Refer to lab manual for results interpretation (as positive or negative)
Biochemical test results (continued)
Nitrate test (NO3 reduction)
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.
uninoculated
uninoculated
uninoculated
after incubation
after incubation,
Reagents added
Urea hydrolysis test
after incubation
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).
Bactria that produce urease, hydrolyze urea to ammonia (NH3). This is indicated by the development of hot pink color.
Biochemical test results (continued)
Phenol red (Carbohydrate) broths
Glucose
Lactose
Sucrose
Mannitol
Gelatin agar deep (hydrolysis)
uninoculated
uninoculated
After inoculation/incubation,
mannitol, sucrose, lactose, and glucose broths look like tube on the right hand.
after incubation
Bacteria that produce gelatinase, hydrolyze gelatin in gelatin deep. Liquefied gelatin/hydrolyzed 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.
Refer to lab manual for results interpretation (as +/-)
Biochemical test results (continued)
Decarboxylase broths
Arginine
Lysine
Ornithine
Deaminase (phenylalanine)
uninoculated
uninoculated
after incubation,
with reagent
After inoculation/incubation:
lysine, ornithine, arginine
from left to right
Refer to lab manual for results interpretation (as +/-)
Biochemical test results (continued)
Casein
Starch
Refer to lab manual for results interpretation (as +/-)
uninoculated
uninoculated
after incubation,
Reagent added
after incubation
after incubation
uninoculated
Lipid
ONPG (Beta galactosidase)
DNAse
Biochemical test results (continued)
uninoculated
uninoculated
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
On a clean slide, mix a loop-ful of your unknown with a hydrogen peroxide (H2O2). Bubbles formation is positive for an enzyme called catalase.
On a piece of blotting paper add a drop of oxidase reagent. Smear oxidase wet area with your unknown. Blue color formation within 20 seconds is an indication of oxidase production by your unknown.
inoculated
Using a straight wire inoculate 3 TTC deeps (semi-solid media) with your unknown by stabbing from center top to down. Incubate overnight @25/37deg C and check
where bacteria are growing. If unknown grows following stab-line, it is non-motile.
(negative). If growing in the entire medium, it is motile (positive). Motility test
