As mentioned in Exercise 8, “Identifying Gram Negative Rods”, identifying bacteria is a common activity in the microbiology lab. Like the game Clue™, each time you gather a piece of information to solve the mystery, you gather some information that supports some identities and eliminates others from contention. In the lab, the process continues as you gather more information until only one microbe remains and all others have been eliminated as possibilities. Thus, identification of microbes is a process of elimination based on logic and carefully-performed tests to determine the capabilities of the unknown microbe.
The pieces of evidence used in the microbiology lab are your observations of the shape, morphology, Gram reaction, and the results from biochemical tests performed using a pure culture of an unknown organism. Results are compared to tables of known results for all bacteria with similar shape, morphology and Gram reaction to see where similarities and differences occur. Such tables in VirtualUnknown™ Microbiology can be accessed by clicking “Identification Matrix” under the “View” option in the lab. Displayed will be tests along the left and the microbes still possible (based on the results you have input) across the top. Every time you record a test result, the Identification Matrix shrinks as the columns with results that directly conflict with those you record for your unknown are eliminated from the table. Those microbes with results that agree with those you record (or that are displayed as [+], d, or [-]) remain possibilities and more testing is needed.
A way of gauging how much progress is being made is to observe the Virtual Lab Report to see how each test result reported increases the number of microbes eliminated. Ideally, every test conducted would reduce the number of possible microbe IDs in half (just as each round of the “March Madness” basketball tournament reduces the number of possible national champions in half). Smart testing reduces the number of tests needed to provide an identification.
The job of the microbiologist is to determine which tests are useful for skillfully reducing the number of possible identities until only a single name remains. That name is the identity of the unknown – the only microbe with test results identical to those you recorded.
Open the Virtual Lab and obtain a new unknown. Select “Gram Positive Coccus” from the Exercise Topic or Subgroup dropdown box and provide the identifier “gramposcoccus”. Read the Case Study and record the Gram stain results.
Move the Virtual Lab to the right side of your screen.
Select the Identification Matrix from the View dropdown box. The matrix will fill the screen. In the upper right is an icon allowing you to either resize or minimize the ID Matrix to the tray below the lab. Resize the matrix and place it to the left of the Virtual Lab. Your setup should look like this:
It is not possible to see all tests or organisms in the matrix at this point – too many columns, too many rows! However, the number of possible IDs for your unknown microbe can be reduced significantly through the results of a few standard tests that are the starting point when tackling the identification of Gram positive cocci. As you record results for these tests in the Virtual Lab, you will quickly see columns disappear as bacteria with conflicting results are eliminated from the matrix. These basic tests leave you with a manageable number of unknowns that can be pared down by a few obvious tests to give you a definitive ID. Consulting the ID Matrix and Help files to learn how to perform the tests gives you a fair shot at completing the ID quickly and easily.
A good starting point would be to use the Help Files, particularly the “Show Me How To…” files related to identify bacteria. Work through the Help files and you will find a recommended approach to get started on the ID process for identifying your Gram positive coccus. What tests would you perform initially to start you on your way?
A key “first test” used in a clinical setting is evaluating growth characteristics on 5% sheep blood to detect presence of enzymes that destroy and digest blood cells and heme. Find the Help file information on blood hemolysis to learn more about this test. What are the three possible results for the test?
Just for fun, click on Unknown and select Record Results. We are going to assign results without doing the tests. Find Alpha Hemolysis or Beta Hemolysis and record a result for one of these as positive. Watch as the number of columns is reduced by the results you enter. Why would growth characteristics on blood agar be an important start for identifying bacteria in a hospital lab?
Repeat the manual input of test results for the bile esculin slant. Record both as positive. Note the change in the number of columns as these results are entered. Why are positive results with bile esculin important information toward understanding the normal ecology of the organism being tested?
What would be the next test you would perform on the unidentified Gram positive coccus to help provide a quick identification?
Print out your Virtual Lab Report and submit it to your instructor.
In the exercises to come, you will use the same logic with real test results to identify unknown Gram positive cocci.
One of the first tests often conducted with bacteria isolated from a hospital setting is determining the pattern for blood hemolysis. Initial isolation of bacteria is very often accomplished on blood agar to encourage growth of all organisms – many disease-causing organisms are notoriously fastidious and cannot be recovered from clinical samples without the especially rich nutrients supplied by 5% sheep blood added to a basic nutrient agar.
However, differences in abilities for bacteria growing on blood agar result in differences in appearance. Some bacteria possess a hemolytic (“blood splitting”) enzyme that breaks the erythrocytes to release the heme. Colonies for bacteria with this hemolysin produce a discoloration of the agar (often greenish). These bacteria are termed “alpha-hemolytic”, and the result seen in the agar is termed “alpha- hemolysis” (also sometimes shown as “α-hemolysis”).
Other bacteria are capable of lysing erythrocytes AND digesting the heme. The result is clearing of the agar under the colonies as the blood cells and their contents are digested completely. This is termed beta-hemolysis (“β-hemolysis”) and these bacteria are termed beta-hemolytic.
There is a third type of hemolysis termed gamma-hemolysis (“γ-hemolysis”). This term is something of a misnomer, since in gamma-hemolysis, NO change is seen in the agar – no discoloration, no clearing.
In the exercise to follow, you will gain some firsthand experience in streaking blood agar plates (virtually) and interpreting the types of hemolysis that result.
Enter the Virtual Lab and click “New” to obtain a new unknown. Select “Blood Hemolysis 1” from the “Exercise Topic and Subgroup” list. Give the identifier “hemolysis1”
Select 5% sheep blood agar from the list of media. Label the plate “ba”.
A plate of blood agar and a tube of the unknown culture appear in the lab. Use the tube-to-plate aseptic technique for preparing isolated colonies and streak the plate, as shown in an earlier exercise. A plate with streak marks (as shown at right) will appear. Be sure the traffic signals indicate no contamination. If so, discard the medium and get a fresh plate to start over.
Place the plate in the incubator and click “New Day” to give the plate 24 hrs
of growth. Retrieve the plate and right click to “Record Results”. A closeup of the plate appears.
Using the information provided above (and elsewhere if so instructed) record the type of hemolysis present in the software and here (Circle):
Discard the plate. Click the “New” button and repeat this process with the “Blood Hemolysis 2” unknown from the “Exercise Topic and Subgroup” list. Give the identifier “hemolysis2”.
Streak the plate and incubate it at 37C overnight.
Retrieve the plate and right click to “Record Results”. Are the halos transparent or a discoloration of the medium?
Record your results in the software and here (Circle).
Use the Help files and Identification Matrix to provide answers to the following…
Which form of hemolysis is most frequently associated with these microbes?
Staphylococcus aureus:Streptococcus pyogenes:
Streptococcus pneumonia:Streptococcus mutans:
Enterococcus faecalis:Staphylococcus epidermidis:
How is blood agar related to chocolate agar?
How might the presence of hemolysins enable a microbe to be more virulent than one without hemolysins?
Dispose of any media before exiting the Virtual Lab.
The catalase test is traditionally one of the first test performed on Gram positive cocci. It provides a quick means for distinguishing between major groups: Staphylococcus and Micrococcus (catalase positive) and the Streptococci – Streptococcus, Enterococcus, and Lactococcus spp. (catalase negative).
Catalase is a protective enzyme produced by many aerobic bacteria. The enzyme breaks down hydrogen peroxide produced during respiration. You may recall that the cytochromes pass along electrons to a final electron acceptor (FEA). In aerobic respiration that FEA is oxygen, in a process that adds two electrons and two protons to ½ O2 to make water. However, if only half the diatomic oxygen (O2) is used, that leaves a very dangerous substance around – singlet oxygen (O-). Singlet oxygen is very reactive and often reacts with 2322 2
diatomic oxygen (O ) to make ozone (O -) or with water (H O) to make hydrogen peroxide (H O ). All three substances – singlet oxygen, ozone, and hydrogen peroxide – are very toxic to cells and can disrupt metabolism or cause catastrophic mutations. Vitamins and dietary supplements often are advertised to be “free radical scavengers” to accomplish the removal of these and other reactive substances from our systems.
Aerobes typically have one or more enzymes that detoxify these products of metabolism. Catalase is one of them. It has the ability to convert hydrogen peroxide into diatomic oxygen and water. You may have placed peroxide on a cut and noted the bubbly foam produced as it interacts with catalase found in blood and our cells. It is administered as a liquid, but as the enzyme splits peroxide to generate oxygen gas, bubbles flow out of the liquid.
In the Virtual Lab, the catalase test is not performed, but instead is observed. Below is a description of how it can be accomplished in the software.
Open the Virtual Lab and click the “New” button. Select the “Media Tests 6” unknown from the “Exercise Topic and Subgroup” list. Give the identifier “MT6”. Review the Case Study and record the Gram stain results. Based on shape and grouping, what would you predict to be the genus of this organism?
As done in a previous exercise, open the Identification Matrix and reduce its size to place it side-by-side with the Virtual Lab. This is done so you can see the changed occurring as you conduct the catalase test.
From the “View” list, select “Catalase Video”.
Appearing will be a viewer where you can watch the addition of 3% hydrogen peroxide to a smear of fresh growth on a microscope slide. The video can be replayed if needed. Based on the information provided above, is this organism catalase positive or negative? (Circle)
What evidence supports your interpretation?
What would the video look like if the result was opposite to that portrayed in the video?
Record the results in the software and watch the Identification Matrix for changes. Click on View and select Lab Report to determine how many species of bacteria were unknown based on the result you recorded. How many were eliminated?
Would strict aerobes or strict anaerobes be more likely to possess catalase? Explain your reasoning.
Close the Virtual Lab.
Bile salts are substances produced by the digestive system to emulsify fats, making them digestible in the aqueous environment of the human body. Because the bacterial membrane and Gram negative cell wall also contain fat-like lipid materials, they also are subject to disruption by bile salts. However, some bacteria are resistant to bile salts. Bile esculin agar is used to test the ability of an unknown organism to grow in the presence of bile salts. Enteric organisms, such as Gram negative bacilli typically found in the gut, would be expected to grow on bile esculin. Also growing would be Gram positive cocci such as the enteric cocci – Enterococcus spp. And, some other bacteria such as the Staphylococci often are able to grow on bile.
Also present in the medium is esculin, a glucoside compound. Some bacteria can digest esculin to produce esculetin, which gives the agar a brown-to-black discoloration. Thus, bile esculin agar is useful for two tests – ability to grow on bile, and ability to metabolize esculin to esculetin. A positive result for growth on bile is obvious growth. A positive result for esculin hydrolysis is a very dark discoloration of the medium. Below you will see how this test is performed.
Enter the VirtualUnknown™ Microbiology’s Virtual Lab and select Media Tests 5 from the list of predefined unknowns. Label it MT5. This organism is Staphylococcus aureus. Your instructor will advise you on whether to check the box permitting autoinoculation.
Select Bile Esculin from the Media dropdown list. Inoculate this medium and place in the 37C incubator. Click “Next Day” to complete its 24-hour incubation period.
Remove the medium from the incubator and record the results in the Virtual Lab Report. Also record the results in the table below. If you need help interpreting the test, click on the “M” icon and to read up on how the medium is used. You can look up the tests for which it is used by clicking on the “T” icon and reading up on those tests.
Media Tests 5
Staphylococcus saprophyticus Media Tests 6
Growth on Bile:
Once the test is completed, you may discard the media. Review the Virtual Lab Report and the Identification Matrix to see how the results from the bile esculin agar have reduced the number of possible identities from the original 50+.
Repeat the above assignment using Media Tests 6 (MT6, Staphylococcus saprophyticus) and record the results in both the software AND in the table above.
Use the table, the Help files in VirtualUnknown™ Microbiology, and the information in your textbook and laboratory manual to answer the following questions:
Which of the following bacteria would be most likely to encounter bile salts in their normal habitat? (Circle all correct answers)
Staphylococcus aureus Enterobacter aerogenes Enterococcus faecalis
Why would those you selected be expected to grow on bile esculin agar?
Where in the digestive system are lipids digested? Is there any evidence that these bacteria are more inclined to cause infections in those organs than are bacteria incapable of growing on bile?
It is possible to have an organism that is positive for both “growth on bile” and “esculin hydrolysis”, an organism that is negative for both, and an organism that is positive for one but not the other. Explain the only possible way an organism could be positive for one but not for the other.
Print out your lab reports and attach them to this exercise before submitting to your instructor.
MANNITOL SALT AGAR
Often, the first step taken to isolate a Gram positive coccus is the use of Mannitol Salt Agar (MSA). MSA is a nutrient agar that contains a few additional substances. It contains the sugar mannitol and the pH indicator phenol red to show whether the sugar has been used to produce acids. It also contains 7.5% sodium chloride (NaCl) as an inhibitor to prevent the growth of unwanted bacteria. Thus, it represents a medium that is both selective (because of the salt) and differential (because phenol red reveals whether an unknown organism has the ability to use mannitol).
Use of salt is one of the oldest methods for preserving foods. The salt draws water out of spoilage organisms, stopping their metabolism. This is proof of the saying that “water follows salt” and is an excellent example of osmosis in action – water leaving cells to dilute the salt in the surrounding vicinity.Some bacteria are very resistant to drying due to modifications in their cell membranes. Among them are many skin bacteria.
An unknown specimen thought to contain such bacteria is streaked for isolation on MSA. Those that form colonies will either turn the medium yellow underneath (if mannitol is fermented) or hot pink (if mannitol is not fermented, the non-sugar nutrients used yield alkaline products). Thus, two tests are conducted using MSA: (1) growth of the unknown in the presence of 7.5% NaCl, and if growth occurs (2) whether the microbe can ferment mannitol. Below is some virtual practice in the use of the medium.
Open the Virtual Lab and click “New”.
From the dropdown list of unknowns, select “Media Test 5”. This organism is Staphylococcus aureus.
Read through the Case Study and interpret the Gram stain. Once in the Virtual Lab, select Mannitol Salt Agar from the Media list. Label the plate “msa”.
Inoculate the plate following the tube-to- plate streak process for producing isolated colonies. Incubate overnight at 37C.
Remove the culture from the incubator and observe the growth. Provide results for these two tests:
Growth on 7.5% NaCl: + –
What result would be expected in the “growth in 6.5% NaCl test” for bacteria capable of growth on mannitol salt agar? Explain.
Dispose of all media and close the Virtual Lab.