Practical Exam #2: Review

I. Labs / lectures 15 through 21

A. Medical microbiology is a branch of microbiology that investigates pathogenic microorganisms. This field focuses on the isolation and identification of organisms using staining and biochemical techniques.

1. Isolation - Before an organism can be identified, it must be obtained in pure culture. After taking a patient sample (urine, saliva etc......), selective and differential media are generally used to isolate the organism of interest.
2. Gram staining - ALWAYS an IMPORTANT first step- determines Gram reaction, morphology and arrangement.
3. Biochemical tests - Suggestion: begin by reviewing your lecture notes (lectures 15 through 21). The important aspects of each test are then stressed in the Summary of Biochemical Tests found on the internet. Focus your study here. If given 2 bacterial isolates, be certain that you can ID the one that is catalase positive.
4. Microorganisms with which to be familiar:

Staphylococcus spp:
S. epidermidis
S. saprophyticus
S. aureus

Micrococcus spp.
Micrococcus luteus

Streptococcus spp:
S. pyogenes
S. pneumoniae
S. mitis
S. agalactiae
S. mutans

Enterococcus spp.
Enterococcus faecalis
Enterococcus faecium

Enterobacteriacae
Escherichia coli
Shigella dysenteriae
Proteus mirabilis
Salmonella typhi
Klebsiella pneumoniae

Pseudomonas
P. aeruginosa

Other
Bacillus subtilis (as discussed in lab 21)

*Be familiar with the definitive biochemical tests used to identify these organisms. For example, the urease test and the SIM are important tests when trying to identify Proteus mirabilis. Most of this is included in the Searchable Summary of Microorganisms.
Practice 1: An MSA plate is streaked with an unkown organism and incubated for 24 h. After the incubation, the plate is bright yellow. Is the unknown organsim capable of fermenting mannitol? Give an example of an organism that would be incapable of growing on an MSA plate.
Practice 2: If a student inoculated a coagulase tube with Staphylococcus aureus, what would the student likely see after incubation?
Practice 3: Upon performing the oxidase test on two unkown bacteria (A and B) a student notes that bacterium A immediately turns bright purple and bacterium B turns white. Which bacterium (A or B) is more likely Pseudomonas aeruginosa?
5. Be sure that you are familiar with how to use the dichotomous keys. If given a set of test results, be able to use one of these keys to identify the implied organism.

B. Reserve and working stock cultures

1. The working stock was the stock that we used throughout the identification process as an inoculum source. This stock was a slant tube inoculated with an isolated colony by making several passes across the agar surface.
2. The reserve stock served only as a backup in case of contamination of the working stock. This was a stab tube and was inoculated with a single colony by stabbing a needle 1 time to the bottom of the tube.
Using a single stab restricts growth and limits nutrient depletion and toxic waste accumulation.
3. Be able to aseptically inoculate a reserve or working stock.

C. Preparation of wine

1. A must is juice from anything that can be fermented.
2. Why ferment even though it builds up a toxin (ethanol) ? - Allows the yeast to recycle reducing power in the form of NAD+ and thus continue to produce ENERGY via glycolysis!
3. Wine fermentation

a. Performed by yeast (Saccharomyces cerevisae var. ellipsoides).
b. Fruit sugars are broken down to give acetaldehyde and then alcohol.
c. The concentration of alcohol is a function of the amount of sugar and the tolerance of the yeast.
d. What things affect the taste of wine??
e. Be able to read a hydrometer!

D. Visual clues such as colony morphology and nutrient agar growth patterns can aid in the identification of unknowns.
Be able to recognize the technical terms used to describe colony shape, margin, elevation, optical properties and texture. Also be familiar with the terms used to describe the growth patterns in nutrient agar. (*Practice: Which one of the following terms might be used to describe colony shape? Raised, Dull, Lobate or Circular)
See also colony morphology, broth growth patterns and pigment production under additional information on the course web page.

III. Lab / lecture 22: Immunology

A. Immunology is the study of defensive responses to foreign invaders (called antigens). Defense responses can be either nonspecific or specific. Nonspecific responses are innate and constantly operating (all brawn and no brain). Specific responses develop after nonspecific responses and are directed specifically at the antigen (the brains of the operation).
Practice: Which one of the following describes a specific immune response?
a. fever
b. inflammation
c. the phagocytic action of nuetrophils and macrophages
d. Secretion of an antibody by a B-lymphocyte
B. The specific immune responses are divided into either humoral or cell-mediated responses. Humoral immune responses involve the production of antibodies by B-lymphocytes and the cell-mediated immune response involves the destruction of "self cells gone wrong" (tumor cells, parasitized cells, transplanted cells) by T-lymphocytes.
We focused on the humoral immune response and specifically antibody/antigen interactions. Remember that an antibody is a glycoprotein that reacts specifically with a particular antigen and tags it for removal or destruction!
Which statement/s about an antibody is/are true?
a. Antibodies are bivalent, Y-shaped molecules.
b. Antibodies are composed of two heavy chains and two light chains bound by disulfide linkages.
c. Antibodies can react with multivalent antigens to form a lattice network.
d. All of the above
C . Serology is the science of detecting and measuring antigen-antibody reactions.

1. Precipitation - A bivalent antibody reacts with a multivalent antigen that is soluble (e.g. a protein or carbohydrate) and forms a lattice network of alternating Ab-Ag. A precipitation was formed in the Ouchterlony gel diffusion experiment.
2. Agglutination - A bivalent antibody reacts with a multivalent antigen that is insoluble: particulate or cellular (e.g. bacteria, viruses, tumor cells, foreign red blood cells etc..) and forms a lattice network of alternating Ab-Ag. An agglutination is demonstrated by hemagglutination seen in blood typing.

D. Blood typing

1. Type A blood cells, have on their surface, a type A antigen. If antibody-A is added to type A blood => hemagglutination. A person who has type A blood sees type B antigens as “non self”. If a type A person receives blood from a type B person, the type A person will recognize the type B blood as foreign => hemagglutination (rejection).
2. A person with type AB blood has both A and B antigens on his/her cell surfaces and thus sees everything as “self” (universal acceptor).
3. A person with type O blood has no antigens on its surface. A person of any blood type can be given type O blood because there is nothing on the cells to be recognized as foreign (universal donor). However, a type O person can only accept type O blood, why?

E. Rh factor

1. This refers to a protein found on the surface of red blood cells called RhD. Some people have it (Rh+) and some don’t (Rh-). A person who has type A blood and is Rh+, has A+ blood.
2. If an Rh- woman is carrying a child of an Rh+ man, after the first pregnancy she may begin to produce antibodies to the Rh factor. If not treated with Rhogam than this can be a problem in subsequent pregnancies, as the mother may produce antibodies against the unborn child if it is Rh+.

F. Application

1. Be able to determine the blood type of a person by looking at a blood typeing tray in which the blood has been mixed with antibodies to A, B and Rh antigens. Be able to determine what type of blood would be given to this person if he/she was in need of a transfusion. (Practice: If a drop of blood is added to both a drop of antibody A and a drop of Antibody B and hemagglutination is observed only with the Antibody A, what is the blood type?)
2. Be able to read an ouchterlony gel diffusion plate.

G. White blood cells

-Know these in order of abundance, know what they do and whether they are granulocytes or agranulocytes (granulocytes end in -ils (neutrophils, eosinophils and basophils)). When viewing a blood slide, be able to identify the more easily identifiable WBCs (e.g. monocytes and lymphocytes.)
G. Red blood cells
- function in transport of oxygen and carbon dioxide gases. Shaped to fit their function.

 

IV. Lab / lecture 23: Water Microbiology

A. Water microbiology is the bacteriological examination of water to ensure that enteric pathogens are not present in drinking water and presenting a risk of disease (typhoid fever, cholera, amoebic and bacillary dysentery...).
B. Generally it’s not practical to screen for enteric pathogens, so instead we screen for indicator organisms, most commonly coliforms. (Practice: Why is it impractical to screen for enteric pathogens and why is easier to screen for coliforms??)
C. All coliforms are normal flora bacteria that are present in the GI tract of every warm blooded animal. Coliforms are Gram-negative, non-spore forming rods that are aerobic or facultatively anaerobic. All are lactose fermenters.

1. To simply determine if coliforms are present, the presence absence test (PA test) can be used. This is a qualitative test that can detect a single coliform in a 100 mL water sample. The broth contains lactose and a pH indicator. What color does this pH indicator change if lactose is fermented and acid is produced??
2. To determine the number (quantitative) of coliforms present, the membrane filtration method can be used. Endo medium inhibits the growth of Gram-positive organisms (What selective ingredients do this??). It also differentiates between coliforms that ferment lactose (red or dark pink colonies) and those that don’t (light, colorless colonies). Be sure to understand how to take into account dilution factors (see data table in the lab notebook).
3. The multiple tube fermentation method also determines, quantitatively, how many coliforms are present. This test has three stages. The first stage is the quantitative stage, the other two are merely confirming that a coliform has been identified.

a. Stage #1: The presumptive test

1.) Three sets of three lactose broth tubes are inoculated with either 0.1 mL, 1 mL or 10 mL aliquots of the sample.
2.) Know how to interpret, determine a profile and use the MPN table. Realize that this is less accurate than the membrane filtration method. Why?

b. Stage #2: The confirmed test
- An EMB plate is streaked from one of the lactose broth tubes showing a positive result. (Practice: A colony on an EMB plate appears dark green and has a metallic sheen, is it positive or negative for lactose fermentation. Does this confirm the initial lactose broth result (the tube was bright yellow)?)
c. Stage #3: The completed test
- Inoculate another lactose broth tube.
- verify Gram-negative and non-spore forming using staining methods.

D. Not all coliforms are fecal. To differentiate between fecal and nonfecal coliforms the IMViC test is used. (Practice: What tests does IMViC stand for and what would be the test results for a fecal coliform?)

E. What must be true in order for water to be considered potable??

V. Lab / lecture 24: Soil microbiology

A. There is a plethora of microorganisms in the soil. We isolated and discussed the following.

1. Bacteria - isolated using nutrient agar. Are these bacteria more likely to be Gram-positive or Gram-negative? Give an example of one Gram-positve and one Gram-negative bacterium that would likely be found in a soil sample.
2. Streptomyces

a. belongs to the bacterial group called Actinomycetes.
b. has filamentous growth (looks like a fungus but is not).
c. Produce geosmin, a compound that gives soils their earthy odor.
d . is a source of antibiotics (e.g. streptomycin)
e . can be isolated using Jensen’s molten agar.

3. Molds -

a. Generally found in well-aerated soils and most are saprophytes. Often capable of digesting complex polysaccharides such as cellulose.
b. Selected for using Martin’s molten agar. This agar contains two selective ingredients: Rose Bengal dye and streptomycin that inhibit the growth of non-mold soil organisms.

4. Nitrogen fixing bacteria

a. Nitrogen is essential to life (e.g a building block for amino acids). However, most organisms cannot utilize N₂ gas as a nitrogen source. Nitrogen fixing prokaryotes convert N₂ gas into more usable forms of nitrogen such as ammonium and nitrate. KNOW THE NITROGEN CYCLE!!
b. Azotobacter and Rhizobium were the two N₂ fixing genera discussed in class. Rhizobium is a symbiotic organism of root nodules but Azotobacter is free living which is why we were able to isolate it from soil using nitrogen-free media. [What interesting things about Azotobacter were noted on the Gram stain?]

B. Other things of note

1. Be able to recognize Martin’s molten agar and nitrogen-free agar.
2. Understand the dilutions and the calculations of titer and number of organisms per gram of soil (see below under general review.)

VI. Lab / lecture 25: Food Microbiology

A. Microbes are important in food production: beer, wine, cheese, yogurt, sour cream, buttermilk etc... Lactococcus is used in the production of hard cheese and Lactobacillus in the production of soft cheese. Streptococcus is used to make sour cream and yogurt and Acetobacter is used in the production of vinegar.
B. Some microorganisms are used as food preservatives (i.e. LAB secrete lactic acid and often produce bacteriocins.)
C. On the down side, microorganisms cause food spoilage and disease. The two types of food-borne disease are food infections and food intoxications. Practice: Is botulism considered a food intoxication of a food infection? Why?
D. Antimicrobial substances are often found naturally in food. Practice: List two types of food that contain antimicrobial compounds. What is the mechanism of action of each one of these compounds?
E . Snyder's Test for Susceptibility of Dental Caries

1. Used to detect the amount of Lactobacillus present in saliva.
2. Attempts to positively correlate the amount of Lactobacillus with susceptibility to cavities.

VII. Lab / lecture 26: Preparation of yogurt

A. A majority of this lecture was for fun, however there are some important points:

1. Lactic acid bacteria (LAB) perform dairy fermentations. They do so by fermenting the milk sugar, lactose, to lactic acid. This lowers the pH of the milk, inhibiting the growth of acid-intolerant microbes, while at the same time providing products with various tastes, textures and aromas.
2. Know how yogurt is made (e.g. pasteurization followed by cooling and then inoculated with a 1:1 ratio of Streptococcus thermophilis and Lactobacillus bulgaricus etc......)

VIII. General Information

A. Sterile technique
-Although this was introduced in the first half of the semester, it continues to be an essential part of every procedure. As in practical exam #1, be able to demonstrate competency with sterile pipetting and streaking.
B. Pipetmen

1. Be able to use a p20, p200 and p1000.
2. Be able to determine what a volume indicator will look like in order to pipet a specified volume. Practice: draw the volume indicator of a p200 set to aliquot 145 mL.
3. BE ABLE TO CONVERT from milliliters to microliters and vise-versa.

C. Dilution problems

1. Review lecture 5 and procedure 5 involving dilutions. This is not necessary if you feel you are still competent with dilution problems.
2. **Review the dilution problems involved in labs 24 and 25.