LAB 10 – Biochemical Bacterial Identification 2
Introduction
In the previous lab, we used biochemical tests to differentiate between different species of gram-negative bacteria. In this lab, we will be looking at other biochemical tests that can differentiate different Gram-positive cocci.
There is no separate worksheet. Answer the questions in each section as you work through the labs. This lab is worth 10 points.
Part 1: Catalase and Coagulase tests
Do the following lab:
https://vlab.amrita.edu/?sub=3&brch=73&sim=703&cnt=1
Catalase is an enzyme, which is produced by microorganisms that live in oxygenated environments to neutralize toxic forms of oxygen metabolites; H2O2. The catalase enzyme neutralizes the bactericidal effects of hydrogen peroxide and protects them. Anaerobes generally lack the catalase enzyme. Catalase mediates the breakdown of hydrogen peroxide H2O2 into oxygen and water. To find out if a particular bacterial isolate is able to produce catalase enzyme, small inoculum of bacterial isolate is mixed into hydrogen peroxide solution (3%) and is observed for the rapid elaboration of oxygen bubbles occurs. The lack of catalase is evident by a lack of or weak bubble production. Catalase-positive bacteria include strict aerobes as well as facultative anaerobes. They all have the ability to respire using oxygen as a terminal electron acceptor.
The catalase test is primarily used to distinguish among Gram-positive cocci: members of the genus Staphylococcus are catalase-positive, and members of the genera Streptococcus and Enterococcus are catalase-negative.
Coagulase is an enzyme produced by S. aureus that converts (soluble) fibrinogen in blood plasma to (insoluble) fibrin. The fibrin clot may protect the bacterium from phagocytosis and isolate it from other defenses of the host. The fibrin coat can, therefore, make the bacteria more virulent. This test is used to distinguish between different types of Staphylococcus isolates: Staphylococcus aureus (positive) from Coagulase Negative Staphylococcus (CONS).
- (0.5 pts) A positive result in the catalase test means that the bacterial species is capable of doing what?
The bacterial species is capable of breaking down the peroxide into water and oxygen.
- (0.5 pts) The purpose of the catalase test is to distinguish which 2 classes of microorganisms apart from each other?
Catalase test is used to differentiate the catalase positive bacterial species such as Staphylococci from the catalase negative bacteria species such as streptococcus.
- (0.5 pts) What is the function of the Coagulase enzyme?
The coagulase enzyme is responsible for forming a clot which may protect the bacterium from phagocytosis aureus (positive) and isolate it from another defense of the host. It clots the blood by converting the soluble fibrinogen to fibrin.
- (0.5 pts) The purpose of the coagulase test is to distinguish which 2 classes of microorganisms apart from each other?
The purpose of coagulase test is to differentiate the species of the genus Staphylococci into two groups; Coagulase positive Staphylococci and Coagulase Negative Staphylococci.
Part 2: Streptococcus Identification
Streptococcus is a genus of coccus Gram-positive bacteria. Most streptococci are oxidase-negative and catalase-negative, and many are facultative anaerobes.
For the first 3 experiments, we will be using the Virtual Interactive Bacteriology Laboratory from Michigan State University. http://learn.chm.msu.edu/vibl/
Experiment 1: Hemolysis
Alpha-hemolytic species oxidize the iron in the hemoglobin turning it dark green in culture. Beta-hemolytic species completely rupture the red blood cells creating a visible halo in culture.
Procedure: Click the “Differential Media” button on the left side panel. Scroll down to the sheep blood agar and click on “examples”. You can click on an individual plate to enlarge it. Observe the results and note them in the following table.
| Table 1: (1 pts) | Streptococcus pneumonia | Streptococcus pyogenes (and Streptococcus agalactiae) | Escherichia coli (and Enterococcus faecalis) | Neisseria gonorrhoeae |
| Hemolysis
|
alpha-hemolysis (green) | Beta hemolysis. | gamma-hemolysis | Beta hemolysis |
- (1 pts) In your own words, explain what hemolysis means.
Hemolysis is the breakdown of the red blood cells in the body and this results into the release of hemoglobin into the blood plasma.
Experiment 2: Lancefield grouping
Groups catalase-negative, coagulase-negative bacteria based on the carbohydrate composition of bacterial antigens found on their cell walls.
Streptococcus pyogenes harbors the Lancefield group A antigen, is often called group A streptococcus (GAS), and when grown on blood agar typically produces small zones of beta-hemolysis—a complete destruction of red blood cells.
Streptococcus agalactiae is the species designation for streptococci belonging to group B of the Lancefield classification. In general, GBS is a harmless commensal bacterium being part of the human microbiota colonizing the gastrointestinal and genitourinary tract of up to 30% of healthy human adults.
Streptococcus pneumoniae was recognized as a major cause of pneumonia in the late 19th century. It has no Lancefield group.
Enterococcus faecalis, carrying Lancefield group D antigens, are commensal bacterium inhabiting the gastrointestinal tracts of humans and other mammals.
Procedure: Place your mouse over the “Biochemical Tests” button on the left side panel and choose “Latex Agglutination Test”. Open the module instructions and a new tab should pop up. Go back to the first tab and open the module itself. Follow the instructions and conduct the experiment. Answer the following questions:
- (1 pts) What are in the blue bottles labeled A, B, C, E, F?
- Rhamnose-N- acetylglusamine
- Rhamnose-glucosamine polysaccharide
- Rhamnose-N-acetylglucosamine
- Control
- Glucosamine-N-acetyl galactosamine.
- (1 pts) What is the purpose of the two white “control” bottles?
They present the non-Lancefield pathogens.
- (1 pts) What causes the observed agglutination?
Incorrect reaction between the antibodies and the antigens in the bottles resulting into the clumping up of the erythrocytes and they stick together causing them to agglutinate.
Experiment 3: CAMP test for identifying Group B streptococci
Streptococcus agalactiae secrets a CAMP factor that enlarges the area of hemolysis formed by β-hemolysin from Staphylococcus aureus. You can use this test to distinguish Streptococcus agalactiae from another Streptococcus and Enterococcus.
Procedure: Watch this YouTube video https://www.youtube.com/watch?v=hXfm3suhZ8E and fill out the following table.
| Table 2: (1 pts) | Streptococcus pyogenes | Streptococcus agalactiae | Streptococcus pneumonia | Enterococcus faecalis |
| CAMP (positive or negative) | CAMP negative. | CAMP positive. | CAMP negative | No reaction. |
Experiment 4: Enterococcus faecalis identification
Until 1984, E. faecalis was known as Streptococcus faecalis. Normally commensal, these bacteria can cause infection in people when it enters wounds, blood, or urine. People with weakened immune systems are especially at risk and E. faecalis is believed to be one of the top three leading causes of hospital-acquired infection. You can distinguish E. faecalis from other Streptococcus by their reaction in bile esculin agar and 6.5% sodium chloride.
Bile Esculin Agar: is a selective differential agar used to isolate and identify members of the genus Enterococcus. Positive reaction leads to blackened agar.
6.5 % sodium chloride: Salt acts as a selective agent and interferes with membrane permeability and osmotic equilibrium. Enterococcus is tolerant to salt while Streptococcus are not.
Procedure: Watch this YouTube video https://www.youtube.com/watch?v=N24nEkvERPg and fill out the following table.
| Table 3: (2 pts) | Streptococcus pyogenes | Streptococcus agalactiae | Streptococcus pneumonia | Enterococcus faecalis |
| Bile esculin agar (positive or negative) | Negative | Negative | Negative | positive |
| 6.5 % sodium
Chloride (growth or no growth) |
No growth | No growth | No growth | Growth occurred |