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In many distinct areas of microbiology, the ability to identify microorganisms has important application. For example, in food microbiology it is important to be able to accurately identify food spoilage contaminants. In microbial ecology, the identification of microorganisms helps us characterize biodiversity. In the field of medical microbiology, a branch of microbiology that investigates pathogenic microorganisms, the primary focus is to isolate, identify, and study microorganisms responsible for infectious disease.
Many microorganisms are permanent residents, or normal flora, of the human body. Bacteria that are normal flora are important symbionts of the human body, most of which cause no ill effects and some, which are actually beneficial to human health. Only a small percentage, less than 10%, of all known bacteria are pathogenic, or able to cause disease in a susceptible host. In order to identify an unknown in the clinical laboratory, a sample must be collected from the patient. This could be a sample of urine, feces, saliva, or a swab of the throat or skin. Because the clinical samples will most likely contain many microorganisms, both normal flora and pathogens, it is important to isolate the pathogen in a pure culture using various types of selective and differential media. Following isolation, one of the first steps in identifying a bacterial isolate is the Gram stain, which allows for the determination of the Gram reaction, morphology, and arrangement of the organism. Although this information provides a few good clues, it does not allow us to determine the species or even genus of the organism with certainty. Thus, microbiologists use characteristic biochemical activities to more specifically identify bacterial species. A Few Biochemical/Physiological Properties Used for identification of bacteria include: nutrient utilization (carbohydrate utilization, amino acid degradation, lipid degradation), resistance to inhibitory substances (high salt, antibiotics, etc.), enzyme production (catalase, coagulase, hemolysins, etc.) and motility.
This series of lab exercises will introduce many of the physiological characteristics/biochemical activities of bacteria commonly encountered in a clinical microbiology laboratory. Knowledge of these key characteristics will enable the identification of unknown bacterial isolates. It is important to thoroughly understand the basis for each biochemical test and know the key physiological characteristics of the bacterial genera and species presented in these labs.
Note: Labs 8-10 will utilize a number of different media and tests that are described on the course web page Additional Info (Summary of Biochemical Tests).
Blood agar is a rich, non-selective medium that supports the growth of most bacteria.
However, it is differential based on the ability of the organism to produce enzymes
called hemolysins, which lyse red blood cells (RBC). Three patterns of hemolysis can
be observed on a blood agar plate.
1. Alpha ( a ) -hemolysis: incomplete lysis of RBC - Greenish, cloudy zone around the colony.
2. Beta ( b)-hemolysis: complete lysis of RBC - A clear zone with a clean edge around the colony.
3. Gamma ( g)-hemolysis: no lysis of RBC - No change in the blood agar around the colony.
*Further classificantion of hemolysins can be done using the Streak/Stab technique. The surface streak allows us to see beta hemolysis cause by Streptolysin S, where as another Streptolysin O can only lyse RBCs in anaerobic conditions. Thus beta-hemolysis in the stab shows the presence of Streptolysin O. These are both produced by the organisms Streptococcus pyogenes.
Observe your BAP plates for hemolysis patterns.
Beta-hemolysis: complete lysis of red blood cells (clear areas)
Alpha-hemolysis: partial lysis of red blood cells (greenish areas)
Gamma-hemolysis: no lysis of red blood cells
This type of medium is both selective and differential. The MSA will select for organisms such as Staphylococcus species which can live in areas of high salt concentration (plate on the left in the picture below). This is in contrast to Streptococcus species, whose growth is selected against by this high salt agar (plate on the right in the picture below).
The differential ingredient in MSA is the sugar mannitol. Organisms capable of using mannitol as a food source will produce acidic byproducts of fermentation that will lower the pH of the media. The acidity of the media will cause the pH indicator, phenol red, to turn yellow. Staphylococcus aureus is capable of fermenting mannitol (left side of left plate) while Staphylococcus epidermidis is not (right side of left plate).
Performing a T-Streak on Half the Plate
Inoculate the two MSA plates by streaking for isolation on half a plate.
View your results on your MSA plates. If the organism grew they are tolerant of salt (halotolerant). If the agar turns yellow this indicates your organism is capable of fermenting mannitol.
Coagulase is an enzyme produced by Staphylococcus aureus that effectively clots blood plasma. Coagulase positive Staphylococus spp. form a clot around themselves to protect against the host's immune defenses. A positive coagulase test is indicated by solidification of the rabbit plasma following inoculation and incubation. If a gram-positive cocci is catalase positive (a test we will learn later) and presumed to be a staphylococci, the coagulase test is often performed and can differentiate between Staphylococcus aureus (coagulase positive) and Staphylococcus epidermidis (coagulase negative).