Week 2

Monday, May 20, Day 5

Smearing and Staining

Preparing a Bacterial Smear

1.   Write name of bacterium on one end of the slide.

2.     Place a loopful of distilled water in the center of the slide.

3.     Transfer a small amount of culture from the agar surface into the water drop using a sterile loop. Spread the mixture into a thin film. And allow the smear to air dry.

4.     Pass the slide through the flame three times, with the smear side up. The smear is now “heat fixed.”

5.     Add 95% methanol to the dried smear for about one minute and then rinse off with distilled water. 

Preparing a Simple Stain

1.     Cover fixed smear with several drops of stain for the times indicated below:

Stain	Time
Carbolfuchsin	5-10 seconds
Crystal violet	20-30 seconds
Methylene blue	At least 1 minute
Safranin	At least 1 minute

2.     Rinse the slide with water to remove excess stain, blot with bibulous paper.

3.     Examine under microscope using the oil immersion lens.

Preparing a Gram Stain

1.     Place slide with a fixed smear on rack over sink.

2.     Cover smear with crystal violet for 20 seconds.

3.     Rinse the slide with water to remove excess crystal violet.

4.     Cover the smear with Gram’s iodine for one minute.

5.     Rinse the slide with water to remove excess iodine solution.

6.     Decolorize with 95% ethanol/acetone. Hold slide at a 45-degree angle while adding decolorizing reagent drop by drop until color stops running.

7.     Immediately rinse the slide to remove the decolorizing agent.

8.     Cover the smear with safranin for one minute.

9.     Rinse the slide with water to remove excess safranin.

10. Blot with bibulous paper.

11. Examine under microscope using the oil immersion lens.

Preparing a Negative Stain

1.     Place a small drop if nigrosin near one end of a clean slide.

2.     Use a sterilized inoculating loop to transfer a small amount of bacterial from an agar surface or a loopful of broth culture into the nigrosin drop. Mix well within that small diameter.

3.     Touch the short edge of another clean microscope slide at a 30-degree angle in the bacteria-nigrosin drop.

4.     The resulting smear should include a thin film with a feathered edge. Allow to dry completely.

5.     Examine under microscope using the oil immersion lens.

Tuesday, May 21, Day 5

Staining

Preparing an Endospore Stain

1.     Place the slide on a beaker with boiling water.

2.     Place paper on the slide and saturate the paper with malachite green. Stain for 5-6 minutes. Add additional stain as it evaporates as needed.

3.     Use a forceps to remove the slide from the heat. Remove the paper from the slide and place it in the biohazard bad. Allow the slide to cool.

4.     Rinse the slide with water for about 30 seconds.

5.     Cover the smear with safanin for 60-90 seconds and then rinse to remove and excess safranin.

6.     Blot water from the slide with pieces of bibulous paper.

7.     Examine under microscope using the oil immersion lens.

Preparing a Capsule Stain

1.     Prepare a smear of bacteria in nigrosin as described in the procedure for a negative stain.

2.     After allowing the spread smear to air dry, cover it with safranin or crystal violet.

3.     Gently wash off the excess stain. Avoid excess rinsing which will remove much of the smear.

4.     Blot water from the slide with pieces of bibulous paper.

5.     Examine under microscope using the oil immersion lens.

Enzyme-Based Tests for Identifying Bacteria

Starch Hydrolysis Test

1.     Label the bottom of a starch agar plate.

2.     Use aseptic technique to inoculate the starch agar plate by streaking a short line on the agar surface with a sample of bacteria.

3.     Incubate the inoculated plate upside-down at 30-degrees C.

Casein Hydrolysis Test

1.     Label the bottom of a skim milk agar plate.

2.     Use aseptic technique to inoculate the skim milk agar plate by streaking a short line on the agar surface with a sample of bacteria.

3.     Incubate the inoculated plate upside-down at 30-degrees C.

Gelatin Hydrolysis Test

1.     Label a nutrient gelatin deep tube.

2.     Use aseptic technique to inoculate the nutrient gelatin deep tube by stabbing with an inoculating needle with bacteria.

3.     Incubate the inoculated slant at 30-degrees C.

Fat Hydrolysis Test

1.     Label the bottom of a tributyrin agar plate.

2.     Use aseptic technique to inoculate the tributyrin agar plate by streaking a short line on the agar surface with a sample of bacteria.

3.     Incubate the inoculated plate upside-down at 30-degrees C.

Litmus Milk Reaction

1.     Label a litmus milk tube.

2.     Use aseptic technique to inoculate the litmus milk tube by stabbing with an inoculating needle with bacteria.

3.     Incubate the inoculated slant at 30-degrees C.

Motility Testing

1.     Label the motility tube.

2.     Use aseptic technique to inoculate the tube by stabbing the bacteria into the center of the test medium to about half to three-quarters deep.

3.     Incubate at 30-degrees C.

Wednesday, May 22, Day 7

Reviewing Test Results

Starch Hydrolysis Test

·      After incubation, flood the starch agar surface with Gram’s iodine. After waiting 30 seconds to 1 minute, check for a purple-black color to develop that indicated where starch is present in the agar.

·      A clear area around a bacteria growth indicates a positive test for starch hydrolysis. If color appears around the growth, the test is negative.

·      Result: Negative

Casein Hydrolysis Test

·      After incubation, examine the plate for a clear zone around the bacterial growth on the plate. This is a positive test. If the area around the bacteria remains white, the test is negative.

·      Result: Positive

Gelatin Hydrolysis Test

·      Check for liquefaction by tilting the tube slightly. If the medium in the inoculated tube remains liquid, the test for gelatinase is positive if the control tube has solidified.

·      Result: Gelatin; positive

Fat Hydrolysis Test

·      After incubation, examine the plate for a clear area around the bacteria growth. This is a positive test for triglyceride hydrolysis. If no clearing is seen around the growth, the test is negative.

·      Result: Negative

Litmus Milk Reaction

·      After incubation, examine the tube for its litmus milk reaction each day.

·      Result: Negative

Motility Testing

·      After incubation, examine the tube for bacterial growth that spreads out from the stab.

·      Result: Positive; Motile

Utilization of Carbohydrates

Fermentation of Carbohydrates

1.     Label the tubes (Glucose, Lactose, Sucrose, and Maltose)

2.     Use aseptic technique to inoculate the sugar broth tube with bacteria.

3.     Incubate at 30-degrees C.

MR/VP Test

1.     Label the MR/VP liquid tube.

2.     Use aseptic technique to inoculate the MR/VP liquid with bacteria.

3.     Incubate at 30-degrees C.

 Citrate Utilization Test

1.     Label the citrate slant.

2.     Use aseptic technique to inoculate the tube by stabbing the bacteria into the center of the test medium to about half to three-quarters deep.

3.     Incubate at 30-degrees C.

Tryptophan Degradation Test

1.     Label the tryptophan broth tube.

2.     Use aseptic technique to inoculate the tryptophan broth with bacteria.

3.     Incubate at 30-degrees C.

Nitrate Reduction Test

1.     Label the nitrate broth tube.

2.     Use aseptic technique to inoculate the nitrate broth with bacteria.

3.     Incubate at 30-degrees C.

Triple Sugar Iron Agar (TSIA) Test

1.     Label the TSIA slant tube.

2.     Use aseptic technique to inoculate the TSIA slant with bacteria.

3.     Incubate at 30-degrees C.

Urea Hydrolysis Test

1.     Label the Urea broth tube.

2.     Use aseptic technique to inoculate the Urea slant with bacteria.

3.     Incubate at 30-degrees C.

Thursday, May 23, Day 8

Reviewing Test Results

Fermentation of Carbohydrates

·      After incubation, examine the tube for acid production or acid and gas production. A yellow color is a positive test for acid production. An orange or red color is negative. A gas bubble trapped in the Durham tube is a positive test for gas production.

·      Results: Negative for gas production.

Citrate Utilization Test

·      Examine the slant for a change in color and bacterial growth. A blue color is a positive test; the bacteria utilized citrate. Green color is a negative test.

·      Results: Negative

Tryptophan Degradation Test

·      After incubation, add 5 drops of Kovac’s reagent to the culture. A quick appearance of a red layer at the top of the tube is a positive test for the presence of indole. The absence of a red layer is a negative test: tryptophan was not hydrolyzed.

·      Results: Negative; tryptophan was not hydrolyzed.

Nitrate Reduction Test

·      After incubation, add 5 drops of reagent A and 5 drops of reagent of B to the tube. Gently shake the tube to mix the reagents with the broth. If a pink to read color develops in 1-2 minutes, the test is positive for nitrate reduction. If no color change is seen, add a small amount of powderized zinc to the tube. If you do not observe a color change after 10 minutes, the test is positive for nitrate reduction. If you observe a color change to pink or red, the test is negative.

·      Results: Negative; no nitrate reduction.

Triple Sugar Iron Agar (TSIA) Test

·      After incubation, examine the tube for any changes in the color of the butt and slant, for the appearance of gas, and for a black precipitate.

·      Results: Negative

Urea Hydrolysis Test

·      Examine the tube for any color change. The appearance of a bright pink color is a positive test for urease. No change in the color of the medium is a negative test.

·      Result: Negative test for urease.

Friday, May 24, Day 9

Respiration Tests

Oxidase Test

1.     Obtain an agar plate.

2.     Add a few drops of oxidase reagent to the bacterial colony on the agar plate.

3.     If the bacteria turn deep blue to purple, the reaction is positive for oxidase.

·      Result: Negative for oxidase

Thioglycollate Test

1.     Label a tube.

2.     Use aseptic technique to inoculate the tube by stabbing the bacteria into the center of the test medium to about half to three-quarters deep.

3.     Incubate at 30-degrees.

4.     After incubation, check tube. Refer to the following chart:

Growth only in the top of tube	Obligate aerobes: Require oxygen
Growth only in the bottom of tube	Obligate anaerobes: killed by oxygen
Growth in the middle of the tube	Microaerophiles: grow best with reduced oxygen
Groth is everywhere but best at the top and decreases down the tube	Facultative anaerobes: grow best aerobically but do not require it
Growth is pretty much everywhere uniformly	Aerotolerant anaerobes: do not need oxygen but can tolerate it

MR/VP Test

·      After incubation, add 5-6 drops of the methyl red to the tube and gently swirl. A red color is a positive test for the mixed-acid fermentation pathway. An orange or yellow color is a negative test.

·      Results: Negative