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Name: Class: Date:

MEDICAL MICROBIOLOGY

OBJECTIVES

 To understand the methodology for isolating and identifying pathogenic bacteria from selected human samples.

 To learn about three different types of hemolysis produced by bacteria and evaluate a throat culture for hemolysis.

 To understand the principle and procedure of a semi-quantitative streak plate and analyze the results with respect to a simulated urine culture.

 To compare and contrast coliforms with true intestinal pathogens and interpret the results of a stool culture on Kligler Iron agar.

DIRECTIONS

Selective and differential media and biochemical tests have long been used in the diagnosis of disease. This week we will be attempting to identify organisms, both normal and pathogenic, that are commonly found in different body systems. In some cases, a great deal of important information can be gathered by simply culturing patient samples on specific media.

Day 1

I. Bacteria of the pharynx (throat sample): Each student will perform their own throat culture, by rubbing the back of their throat with a sterile cotton swab. Avoid touching the tongue or soft palate. Use the throat swab to streak the first quadrant of a streak plate on a Blood agar plate. Streak the other

quadrants with an inoculating loop, as we have done previously. II. Bacteria of the urogenital tract (urine sample): Since the majority of urinary tract infections (UTIs) are the result of contamination with enteric bacteria, urine cultures are usually streaked onto both Blood agar and MacConkey agar plates. Counts over 10,000 CFU per mL indicate possible infection, while counts over 100,000 indicate a robust infection. Each group 2 students per group) will culture a

synthetic urine sample using the semi quantitative streak method on both a MacConkey and a blood agar plate. For the semi-quantitative streak method, we will be using a 1µL (0.001 mL) loop, not the one that you normally use for inoculations throughout the semester. Using good aseptic technique, dip the loop in the synthetic urine sample and streak once down the middle of the Blood agar plate, then back and forth

across the entire surface of the plate. Repeat this procedure for the MacConkey agar plate, using a second loop-full of synthetic urine sample. III. Bacteria of the GI tract (stool sample): The bacteria commonly found in the digestive tract are grouped into the family Enterobacteriaceae. Bacteria in this family are characterized by being oxidase negative, Gram-negative rods. Within this family bacteria can be grouped even further: into bacteria

that are part of the normal microbiota of the gut (coliforms), and bacteria whose presence in large numbers results in disease (true pathogens). Coliforms, such as Escherichia coli, are commonly identified by their ability to ferment lactose, whereas true pathogens, such as Salmonella, do not ferment lactose.

We will take advantage of the selective and differential nature of two culture media in order to identify the bacteria in our synthetic fecal samples. Experiment is performed in group of 2 students or 3 students (if 3 students are present on one table).

1. Each table has three stool samples. Each group should pick one sample to test, making sure that all samples are tested within that table. Be sure to record which patient you are testing. 2. Use a sterile swab to thoroughly break up your stool sample in the tube, and make sure the culture is well suspended before taking a sample.

3. Inoculate a Kligler Iron Agar slant by dipping the entire inoculation needle into the fecal sample suspension, stabbing into the agar and then streaking gently onto the surface of the slant. Day 2

I. Bacteria of the pharynx (throat sample): Observe your Blood agar throat culture. Record your observations and conclusions in the following table. (2 pts)

Blood Agar Results

Color Description Interpretation/

Conclusion

Control

Colony Type 1

Colony Type 2

II. Bacteria of the urogenital tract (urine sample):

Observe your urine culture on Blood and MacConkey agar plates and answer the following questions.

1. How many colonies (CFUs) did you count on your Blood agar urine culture? (0.5 pt)

2. Calculate the number of CFUs/ml in your urine culture. Show all work. (1 pt)

3. Does your patient have a urinary tract infection? Explain why or why not. (0.5 pt)

4. Describe the grow type and color on the MacConkey agar urine culture. (0.5 pt)

5. Does your patient’s urine sample contain coliforms? Explain why or why not. (1 pt)

III. Bacteria of the GI tract (stool sample): Observe your three stool sample KIA slant cultures. Record your observations and conclusions in the following table. (3 pts)

Source of

Inoculum

KIA Test Results

Color

(Bottom)

Color

(Top)

Bubbles/

Breaks

Interpretation/

Conclusion

Control

1. Which unknown stool samples are likely to be coliforms? Explain the evidence. (1 pt)

2. Use the provided dichotomous key for bacteria of the G.I. tract to identify the bacteria in each unknown stool sample: (0.5 pt) Unknown #1:

Unknown #2: Unknown #3: