Determining your Bacteria for the Unknown Bacteria Report
David J. Sanchez, Ph.D. and Daniel Miranda, Jr., Ph.D.
The purpose of this report is the present an analysis of an unknown bacterial culture and explain to the reader how the unknown’s identity was determined. The format is in another document contained on this website. The information is on how to determine the identity of your unknown bacteria.
Differential Staining in Practice
STEP 1
Differential Stains allow one to distinguish a particular type of bacterial species. Use of these stains is a key step (yet not the entirety) of the process in determining the identity of an unknown or disease causing bacteria.
Gram + bacteria and Gram – bacteria effectively split the bacterial kingdom into two separate categories.
Acid Fast + bacterial cells are indicative of one main bacterial species, Mycobacteria. This is also a Gram + species. Thus no Gram – bacteria is Acid Fast +.
Endospore + bacterials cells are indicative of two potential bacterial species, Bacillus and Clostridium. All Endospore + bacteria are Gram +.
The following flow chart summarizes the use of these three main differential stains:
NOTE: ALL GRAM + BACILLI IN THIS PROJECT ARE ENDOSPORE +
BASED ON YOUR GRAM STAIN GO TO THE SECTION ON
Gram + Cocci Go to Page 3
Gram + Bacilli (All Endospore +) Go to Page 5
Gram – Coccobacilli Go to Page 6
These are the only type of Bacteria you have in your project
Remember, you are trying to get the “best” match, not every test will align and we didn’t do every test, but the best match is the right one.
Use this page only if your bacteria is listed as Gram Positive Cocci:
Step 1:
Determine if your Bacteria fits this test:
Catalase Test
If your Bacteria was + then it will be in either of the two Genus
1) Staphylococcis
2) Micrococcus
Proceed to Step 2A on this page
-OR -
If your Bacteria was – then it will be in either of the two Genus
1) Streptococcus
2) Enterococcus
Proceed to Step 2B on the next page
Step 2A:
Compare the metabolism of the two Genus to determine which Genus/Species is the best match to your metabolism.
Staphylococcus
S. aureus S. epidermidis
Colony Color Yellow White
Gelatinase Positive Negative
Phenol Red Dextrose Yellow Yellow
Phenol Red Lactose Yellow Yellow
Micrococcus
M. agilis M. luteus M. roseus
Colony Red Yellow Pink
Motility + - +
Lactose Use - - -
Gelatinase Use + + -
Citrate Use - - -
Phenol Red Dextrose Yellow or Red Yellow or Red Yellow or Red
Phenol Red Lactose Red Red Red
Step 2B:
Compare the metabolism of the two Species of Bacteria in these different Genus to determine which Genus/Species is the best match to your metabolism.
Streptococcus epidermidis Enterococcus faecalis
Phenol Red Dextrose Yellow Yellow
Phenol Red Lactose Yellow Yellow
Gas No Gas
Phenol Red Sucrose Yellow Yellow
Gelatinase Negative Positive
Use this page only if your bacteria is listed as Gram Positive Baccili:
Compare the metabolism of the two Species of Bacteria in these different Genus to determine which Genus/Species is the best match to your metabolism.
Bacillus
Gram positive, bacilli to coccobacilli, endospore positive
Usually motile and catalase positive
B. megaterium B. cereus B. subtilis
Lactose Use Red Red Red
Sucrose Use Red Yellow Red
Dextrose Use Red Yellow Yellow
Citrate use - - -
Use these Last Pages if you are Gram Negative Coccobacilli:
Step 1:
Determine if your Bacteria fits this test:
In Phenol Red Lactose: Did your bacteria produce gas?
No gas was produced. Then it will be from one of the following two Genus:
1) Serratia
2) Proteus
Use the following table to determine which Genus/Species is your bacteria. If Not, then move to next step.
P. mirabilis P. vulgaris S. marcescens
H2S (Iron) + + -
Citrate + - +
Yes, Gas was produced in Phenol Red Lactose test?
x If your bacteria produced gas then move to Step 2.
Step 2:
Check which set fits best for your bacteria.
S. flexni | E. Arogenes |S. Sonnei |Citrobacter|E. Coli | K. pneumoniae
Gram ------
Citrate - + - + - +
H2S(iron) - - - + - -
Motility - + - + + -
Gelatinase ------
Lactose - + - ? + +
Sucrose - + - ? ? +
Catalase + + + + + +
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