Isolated bacteria must be further identified at genus and species level. The identification of bacteria is required to cure the disease or infection caused by the bacteria by using appropriate antibiotics. Identification is also important for epidemiological purposes.
How to identify bacteria in the laboratory
The isolated bacteria are further processed by one or a few of the following procedures to identify the bacteria
- Motility testing
- Staining of the isolated bacteria
- Serological tests
- Phage typing
- Identification disc testing
- Semiautomated and Automated identification systems
- Molecular techniques
- Biochemical testing
Motility testing is carried out by preparing a wet mount and is then observed under the microscope. Bacterial motility can also be tested by inoculating the bacteria in the semisolid motility medium.
Staining of the isolated bacteria
Staining the bacteria is the first and most important step in the identification of bacteria. The isolated bacteria are stained by different methods depending on the focusing bacteria. Various staining techniques are as follows:
Differentiates bacteria into two types : Gram positive and Gram negative bacteria.
- Gram positive bacteria can be either cocci, bacilli or vibrios. Gram positive pathogenic bacteria include staphylococci, streptococci, pneumococci, etc.
- Gram negative bacteria may be either cocci or bacilli. Gram negative pathogenic bacteria commonly found are E.coli, Klebsiella, Salmonella spp, shigella, etc.
This is performed if one suspects Corynebacterium spp.
This is necessary for spirochetes and other organisms
Acid fast staining: This is done in cases suspected of mycobacterial infection. Example. Tuberculosis, leprosy etc.
The importance of identifying unknown bacteria with staining is followed by the use of different biochemical reagents and tests to get closer to the identification of bacteria.Many microbiology biochemical tests are available for bacterial identification.Few of them have to be performed depending on the bacteria.The most commonly used biochemical tests for identification of bacteria are mentioned below.All the following tests answers the question,” how do biochemical tests help identify unknown bacterial species“
Purpose of catalase test
The catalase test is a biochemical test facilitates the detection of the catalase enzyme in bacteria. It is essential to
differentiate catalase- positive micrococcaceae from catalase- negative streptococcaceae.Although it is primarily useful in differentiating between genera, it is also valuable in speciation of certain gram positives such as Aerococcus urinae( positive) from Aerococcus viridans( negative) and gram negative organisms such as Campylobacter fetus, Campylobacter jejuni and Campylobacter coli( all positive) from other Campylobacter species.
Catalase test Procedure
- Place a microscope slide inside a petri dish and keep the petri dish cover available
- Using a sterile inoculating loop or wooden applicator stick, collect a small amount of organism from a well- isolated 18- to 24-hour colony and place it on the microscope slide.
- Be careful not to take any agar. This is especially important if the colony isolate was grown on agar containing red blood cells.The transfer of red blood cells to the test may result in a false- positive reaction.
- Using a dropper or Pasteur pipette, place 1 drop of 3 percent H2O2 on the organism on the microscope slide. Don’t mix.
- Cover the petri dish immediately with a lid to limit aerosols and observe immediate bubble formation( Oxygen + water= bubbles).
- Observing the formation of bubbles against a dark background improves readability.
Catalase Test Result and Interpretation
|Catalase Reaction||Suspected Organism|
|Catalase positive bacteria||Staphylococcus spp|
|Catalase negative bacteria:||Streptococcus spp|
The coagulase test differentiates Staphylococcus aureus strains from other coagulase- negative species. Staphylococcus aureus strains are able to coagulate plasma in the tube test and produce cell clumps in the slide test.
The coagulase test can be performed with two different procedures : the slide test and the tube test. The slide test is simple, with results within 10 seconds, but it can give false negatives. The tube test is the definitive test, but it can take up to 24 hours to complete.For both tests, clumping or clots of any size indicates a positive response.While Staphylococcus aureus is the most commonly isolated coagulase positive organism, there are several other Staphylococcus species that are positive for coagulase activity.Staphylococcus schleiferi and Staphylococcus lugdunensis may produce positive results in the slide test for bound coagulase, and Staphylococcus schleiferi and Staphylococcus intermedius may produce positive results in the tube coagulase test.
Coagulase test Procedure
- The slide test is performed by preparing a suspension of bacterial cells mixed in a drop of rabbit plasma on a microscope slide.
- If bound coagulase is present in bacterial cells, the presence of plasma causes bacterial cells to clump.
- The clumping occurs because the clumping factor is an adhesive, which causes the cells to bind to fibrinogen in the plasma. This leads to the visible clumping of bacterial cells on the microscope slide.
- The figure below shows the visible clumping of cells on the microscope slide.
The tube coagulase test
- The tube coagulase test is performed by mixing bacterial cells into a larger plasma volume in a small test tube.
- As the bacteria in the plasma multiply, they secrete staphylocoagulase.Staphylocoagulase causes blood coagulation by activating prothrombin.
- Staphylocoagulase adheres to fibrinogen, forming a complex that cleaves fibrinogen into fibrin, bypasses the blood clotting cascade and directly forms a coagulation of fibrin.
- The formation of a clot will be noted for a positive response within 24 hours.
Coagulase Test Result and Interpretation
|Coagulase Reaction||Suspected Organism|
|Coagulase positive bacteria||Staphylococcus aureus|
|Coagulase negative bacteria||Staphylococcus epidermidis, Staphylococcus saprophyticus|
The oxidase test is a biochemical reaction, which tests for the presence of cytochrome oxidase, an enzyme sometimes called indophenol oxidase. In the presence of an organism containing the cytochrome oxidase enzyme, the reduced colorless reagent becomes an oxidized colored product.
Oxidase test procedure Procedure
There are many method variations in the oxidase test. These include, but are not limited to, filter paper testing, filter paper spot testing, direct plate method and test tube method.
Oxidase Filter Paper Test Method procedure
- In 1% Kovács oxidase reagent, soak a small piece of filter paper and let it dry.
- Use a loop and select a well- isolated colony from a fresh( 18- 24-hour) bacterial plate and rub onto treated filter paper.
- Observe color changes.
- Microorganisms are positive for oxidase when the color changes to dark purple within 5 to 10 seconds. Microorganisms are positive for delayed oxidase when the color changes to purple within 60 to 90 seconds. Microorganisms are oxidase negative if the color does not change or if it takes longer than 2 minutes.
Oxidase Test Result and Interpretation
|Oxidase Reaction||Suspected Organism|
|Oxidase positive bacteria||Pseudomonas, Vibrio cholera|
|Oxidase negative bacteria||E. coli, Klebsiella, Salmonella|
The indole test screens for an organism’s ability to degrade amino acid tryptophan and produce indole. It is used as part of the IMViC( indole, MR- Vp Citrate) procedures, a battery of tests designed to distinguish between members of the Enterobacteriaceae family.
Indole test Procedure
- Inoculate the tryptone broth tube with a small amount of pure culture.
- Incubate for 24 to 48 hours at 37 ° C.
- To test indole production, add 5 drops of Kovác ‘s reagent directly to the tube.
A positive indole test is indicated by the formation of a pink to a red color( ” cherry red ring”) in the reagent layer above the medium within seconds after the reagent is added.
If a culture is indole negative, the reagent layer will remain yellow or be slightly cloudy
Indole Test Result and Interpretation
|Oxidase Reaction||Suspected Organism|
|Indole positive bacteria||E. coli, Vibrio cholera|
|Indole negative bacteria||Klebsiella, Salmonella, Shigella spp.|
The citrate test screens a bacterial isolate to use citrate as its carbon and energy source.A positive diagnostic test relies on the generation of alkaline citrate metabolism by- products.The subsequent increase in the pH of the medium is demonstrated by the change in color of the pH indicator.The citrate test is often part of a battery of tests used to detect gram negative pathogens and environmental isolates.
- Use fresh( 16 to 18 hours) pure culture as an inoculation source.
- Choose a single isolated colony and lightly streak the slant surface. A needle is the preferred sampling tool to limit the amount of cell material transferred to the agar slant.
- Avoid using liquid cultures as the source of the inoculum.The use of citrate requires oxygen and therefore screw caps should be loosely placed on the tube if used.
- Incubate for 18 to 48 hours at 35oC(+/- 2oC).
- Some organisms may require up to 7 days of incubation due to their limited growth rate on citrate medium.
Citrate Test Result and Interpretation
Citrate Positive: growth will be visible on the slant surface and the medium will Be an intense Prussian blue. The alkaline carbonates and bicarbonates are produced As by- products of citrate catabolism, the pH of the medium increases to above 7.6, causing the bromothymol blue to change from the original grey
Citrate Negative: trace or no growth will be visible.No color change will occur; the medium will remain the deep green forest color of the uninoculated agar.Only bacteria that can use citrate as the sole source of carbon and energy can grow on the Simmons citrate medium, so that a citrate- negative test culture is virtually indistinguishable from a uninoculated slant.
|Citrate Reaction||Suspected Organism|
|Citrate positive bacteria||Klebsiella spp.|
|Citrate negative bacteria||E. coli.|
The urease test identifies those organisms that are able to hydrolyze urea to produce ammonia and carbon dioxide. It is mainly used to distinguish urease- positive bacteria from other Enterobacteriaceae.
- Use Christensen’s Urea Agar (4, 5)
- Use a heavy inoculum from a pure culture of 18 to 24 hours to streak the entire slanting surface.
- Do not stab the butt as it serves as a color control.
- Incubate tubes at 35oC with loosened caps.
- Observe the slant for a color change at 6 hours, 24 hours and up to 6 days every day.
- Urease production is indicated by a bright pink( fuchsia) color on the slant, which can extend into the butt.
- Note that any degree of rose is considered a positive reaction.
- Prolonged incubation may result in a false- positive test due to protein hydrolysis in the medium.
- To eliminate protein hydrolysis as a cause of a positive test, a control medium without urea should be used.
Proteeae( Proteus spp., Morganella morganii and some Providencia stuartii strains) will quickly produce a strong positive reaction within 1 to 6 hours of incubation.
Delayed- positive organisms( e.g. Klebsiella or Enterobacter) typically produce a weak positive reaction to the slant after 6 hours, but the reaction intensifies and spreads to the butt after prolonged incubation( up to 6 days).
Urease Test Result and Interpretation
|Oxidase Reaction||Suspected Organism|
|Urease positive bacteria||Proteus spp., Morganella morganii|
|Urease negative bacteria||E. coli,|
It is an important step in bacterial identification.It usually involves the detection of antigens by enzymes or fluorescence immunoassays.Serology is also used to confirm identification obtained using other methods of identifying microorganisms.For example, salmonella species identified by biochemical testing are processed for serotyping by slide agglutination.Another example is Vibrio cholerae.
Phage typing is a method used to detect single bacterial strains.It is used to trace the source of infection outbreaks.The viruses that infect bacteria are called bacteriophages( ” phages” for short) and some of them can only infect a single strain of bacteria.These phases are used to identify different strains of bacteria within a single species.
In the agar, a culture of strain is grown and dried.A grid is drawn at the base of the petri dish to mark different regions.Each square of the grid is inoculated by a different phase.The phage drops are allowed to dry and are incubated: the susceptible phage regions show a circular clearing where the bacteria have been lysed and this is used for differentiation.
Kirby Bauer disc diffusion method is primarily aimed at identifying the antibiotic susceptibility of bacteria. It is also helpful to identify some bacteria such as Micrococcus spp, Streptococcus spp, Moraxella spp, etc.
Semi- automated and automated identification systems
The isolated colonies obtained are processed by this system.The system identifies the bacteria and also performs antibiotic susceptibility tests for them.Microscan walkaway system, Vivitek system, Sensititre Gram Negative Auto identification system, Phoenix system are some of the semi- automated and automatic identification systems available for bacterial identification.
Bactec AFB system, Mycobacteria Growth Indicator Tube( MGIT) and MGIT 960 Aresome Automatic Identification Systems for Mycobacterial Identification.
Molecular methods include G+C percent content, DNA- DNA hybridization, and DNA base sequencing.These methods are not routinely used in hospital laboratories.Amplification techniques such as polymerase chain reaction, ligase chain reaction, strand displacement amplification and nucleic acid sequence- based amplification are used in clinical laboratories for direct bacterial detection.For example Neisseria gonorrhea, leptospirosis, etc.