Biochemical Tests for Bacterial Identification

In this blog post, I am sharing information about the standard, conventional biochemical tests and their primary uses in the Microbiology laboratory for identifying pathogens of interest. Some of the biochemical tests mentioned here, for example, catalase test, and oxidase test, assist us in many ways apart from the primary purpose mentioned in this post;

1. Beta-glucuronidase test (MUG Test)

MUG test is used to identify Escherichia coli. Escherichia coli produces the enzyme β-D-glucuronidase, which hydrolyzes β-D-glucopyranosid-uronic derivatives to aglycons and D-glucuronic acid.

Overview of Biochemical tests for differentiating Gram positive cocci
Overview of Biochemical tests for differentiating Gram positive cocci

2. Bacitracin Sensitivity Test

Bacitracin sensitivity test differentiates Streptococcus pyogenes (positive) from other beta-hemolytic streptococci (resistant).

3. Bile solubility test

Bile solubility test is used to differentiate Streptococcus pneumoniae from other alpha-hemolytic streptococci. Bile or a solution of bile salt, such as sodium desoxycholate rapidly lyses pneumococcal colonies.

CAMP test :
A. Streptococcus agalactiae (positive)
B. Streptococcus pyogenes (Negative)
Image Source: ASM

4. CAMP Test

Certain organisms such as Streptococcus agalactiae (Group B streptococci), produce a diffusible extracellular protein (CAMP) factor that acts synergistically with the beta-lysin of Staphylococcus aureus and causes enhanced lysis of RBCs.

5. Catalase test

Catalase test is used to differentiate Staphylococci (catalase-positive) from Streptococci (catalase test negative)

6. Citrate utilization test

To differentiate members of the Enterobacteriaceae family.

7. Coagulase test

Coagualse test is used to identify Staphylococcus aureus. Coagulase test differentiates Staphylococcus aureus (positive) from coagulase-negative staphylococci (CONS), such as S. epidermidis, S. saprophyticus.

8. DNase test

This test is used to determine the ability of an organism to hydrolyze DNA. It is primarily used to identify Staphylococcus aureus.

9. Indole test

This test is used to determine the ability of an organism to split tryptophan to form the compound indole. In a microbiology laboratory, it is used to differentiate gram-negative rods, particularly E. coli.

10. Litmus milk decolorization test

To help identify Enterococcus and some Clostridia, which have the ability to metabolize litmus milk.

11. Lysine Decarboxylase test

To assist in the identification of Salmonella and Shigella

12. Oxidase test

To help identify Neisseria, Pasteurella, Vibrio, and Pseudomonas. This test is used to determine the presence of bacterial cytochrome oxidase.

13. Urease test

Urease test is used to determine the ability of an organism to produce urease (an enzyme) that hydrolyzes urea. This test is done to help identify Proteus, Morganella, Yersinia enterocolitica, and Helicobacter pylori.

References and further reading

  1. Madigan Michael T, Bender, Kelly S, Buckley, Daniel H, Sattley, W. Matthew, & Stahl, David A. (2018). Brock Biology of Microorganisms (15th Edition). Pearson.
  2. Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

36 thoughts on “Biochemical Tests for Bacterial Identification

    1. Pranab Roy
      Thank you for your question. Why you need to differentiate member of Enterobacteriaceae family from that of Rhizobia? I think you never encounter such problem while identifying clinical isolate. Rhizobia are non-fermentative and forms dry, tenacious colonies.

  1. sir i am persuing msc. biochemistry and its my 4th semester . . . can u please suggest me topics for dissertation?

  2. Sir which biochemical test is used for streptococcus bovis, dysgalactiae, Uberis and agalantiae

  3. this site is amazing. thank you so much. this help me a lot in my preparation for ASCP exam. more power and God bless

  4. It’s quite clear to understand the basic idea of biochemical tests and expected results, but I wanted to know the limitations in these tests, specifically for oxidase, catalase. If it possible plz give a link even possible

  5. Thanks for the concise and beautiful write up. Please i’ll like to know the biochemical tests for lipase producing microorganisms. Thank you.

  6. please, what is the best media that support the biochemical tests on the microorganism

  7. please give me detail about the ATCC CULTURE (MICROBIOLOGICS)
    what does it means and how to find out following( if it is on certificate of analysis)
    purity : 0.1% total pellet cfu
    Recovery : >1000 cfu / pellet bacillus subtilis

  8. Pls i need a procedure and method on how to isolate identifu e.coli in uninary track infection patients

  9. Dear sir,

    Pl suggest the biochemical test chart for differentiation of Enterococcus Fecalis , streptococcus agalactia , dysagalactia and Uberis .


    Dr S G Deshmukh

  10. I want you to write on staph aureus strain identification and then summarize genomic library construction.

      1. Dear sir,
        Could you please provide me your contact email ID? I need some clarification regarding my thesis in relavent to microbiology. Please find my mail ID:

    1. Pham Khan, we do not perform such testing in our laboratory but I think, this link will address your query.

  11. I would like to Request you to provide me any practical concerning bacteria culture ,biochemical test , and Gram negative bacteria that have a resistance to drugs. and i wanted to have a certificate professional on that.

    I.m working for National public Health laboratory in South Sudan and we have a challenges in bacteria culture .
    please tell me if you have a branch for training people , to our neighbor country south Sudan , i may joint you soon .

    my best Regards
    Pham Khan

    1. Dear Pham Khan, We have updated blogs about various biochemical tests:, culture media: etc already in microbeonline. Please go through the relevant post, and if you did not find any content or if you have any query, please let us know, we will try to address your query.
      We are happy to be connected with you and we wish you all the best. We are hopeful, you will strengthen the microbiology laboratory facility of National Public Health Laboratory of South Sudan with your expertise and leadership skills in the days to come. We are sorry, we do not have any branch to train laboratory technician but we are always open to address your issues. As you have said, you have challenges in bacterial culture, if it is related to some information gap, we are ready to provide relevant information based on our experience.

  12. Thank you sir, for your enlightenment. Sir i want to know for in case of urine test examination, is there specific parasite that you can found and others that can be found in urine in rare cases?

  13. sir,
    really this information is more helpful for us but if u add the bacterial colony characteristic of important and common bacteria it will more better to understand the diagnosis of disease and find out etiology of disease.

    thank you

  14. I was reading your comment on id of Vibrio and was a bit confused on your suggestion to perform oxidase
    on maconkey agar.I know that times have changed since I had learnt from my Professor then to never
    perform oxidase on maconkey,because of false results.Please correct me,I am still learning.

    1. Dear Yacoob Mohammed,
      Thank you so much for your valuable comment. My understanding is also the same i.e. oxidase test must be performed from sheep blood agar or another medium without a fermentable sugar as acidification of MacConkey agar/TCBS can give false-negative results. I regret the inaccurate information inadvertently shared and will correct it as soon as possible. Thank you once again for helping me to disseminate accurate information to microbiology enthusiasts.

  15. What criteria will you use to pick unique colonies from your plates? Assuming you cannot test ALL of your bacterial isolates for antibiotic production, how will you prioritize the isolates you choose to study in order to increase the likelihood of obtaining an antibiotic producer? What are the qualities of the isolates you have picked for further studying?

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