Wendy's Microbiology: Isolation & Gram Stain Preparation

Introduction:

Since microorganisms live as mixed cultures in nature, we need to be able to isolate them into a pure culture for study. The most common method to do this is the streak plate method. In this lab you will streak 2 colonies from each of the sample plates for isolation. The streak plate method dilutes the sample mechanically by dragging the loop across the agar surface, separating the organisms. The loop is flamed between each region to help dilute each region further.

Bacteria can also be isolated by using specialized media. These help the microbiologist inhibit growth of unwanted organisms or visually identify likely organisms. Selective media contain stubstances to inhibit the growth of 1 group while permitting the growth of others. Columbia CNA agar contains the antibiotics colistin and nalidixic acid to inhibit the growth of Gram negative organisms. It is selective for Gram positive organisms.

Differential media causes an observable color change, differentiating the bacteria that are able to carry out that specific biochemical reaction. Enrichment media enhances the growth of certain organisms. Some media are both selective and differential. EMB (Eosin Methylene Blue) agar is selective for Gram negative organisms and is differential for certain Gram negative enteric bacteria.

Escherichia coli: large blue/black colon, green metallic sheen

Enterobacter & Klebsiella: large, mucoid, pink/purple colonies, no metallic sheen

Salmonella, Shigella, Proteus: large colorless colonies

Shigella: colorless to pink colonies

Isolation using different media:

Select 2 different colonies from each of the finger culture, hair culture, and air culture and streak for isolation. You will prepare a streak for isolation a plate of Columbia CNA agar and EMB agar for each sample colony. For each colony you will streak ½ of a CNA plate and ½ of a EMB plate.

Label your plates and incubate at 37degrees Celsius.

Gram stain preparation:

Bacteria are not easily visualized under a microscope without staining. Stains allow a greater contract between the organism and its background, allow us to identify the morphology (shape, arrangement, Gram reaction), and see flagella, capsules, endospores, etc. Dyes are stains are generally divided into basic and acidic dyes.

The Gram stain is the most common stain used in microbiology, and is used to differentiate between Gram positive and Gram negative bacteria. Gram positive bacteria stain purple, and Gram negative stain pink. The difference in Gram reaction is due to differences in cell wall structure. The Gram positive cell wall is 60-90% peptidoglycan. The Gram negative cell wall only has 2-3 layers of peptidoglycan surrounded by an outer membrane of phospholipids, lipopolysaccharide, lipoprotein, and proteins. Only 10-20% of the Gram negative cell wall is peptidoglycan.

Heat fixation from agar medium:

1. Place ½ a drop of distilled water on a clean slide.

2. Use aseptic technique to transfer a small amount of culture from the agar surface and touch it to the water until it turns cloudy.

3. Burn the remaining bacteria off the loop.

4. Use the loop to spread the suspension over a large portion of the slide for form a thin film, and allow it to completely air dry.

5. Pass the slide film side up through the flame of the burner 3-4 times to heat fix. Be careful not to use too much heat that may distort the organisms. Too much heat may cause a Gram positive organism to stain Gram negative.

Heat fixation from a broth culture:

1. Aseptically place 2-3 loops of the culture on a clean slide.

2. Spread the suspension over the slide and allow to air dry.

3. Use the burner to heat fix as described above.

Gram stain procedure:

1. Stain the bacteria with the basic dye crystal violet.’(1 minute)

2. Cover the smear with Gram’s iodine solution. This allows better retention of the stain by forming an insoluble crystal violet iodine complex. (1 minute)

3. Decolorize the smear with the Gram’s decolorizer, a mixture of ethyl alcohol and acetone. Gram positive bacteria will retain the crystal violet complex while Gram negative are decolorized. (just until purple stops flowing, and wash with water immediately)

4. Counterstain with the basic dye safranin. Gram positive bacteria are not affected by the counterstain because they are already purple. Gram negative bacteria are colorless, and will become directly stained by the safranin. (2 minutes and then wash with water).

Evaluate your skill in streaking for isolation with the sample colonies:

Results: Note how many different colonies it appears from each sample colony you streaked.