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Laboratory 3

Differential Staining

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Three Main Things We are Doing Today

I. Looking at Results from Last Weeks Cultures

II. Learning to use an Inoculation Loop for Streak Plate Method

III. Differential Stains

I. Looking at Results from Last Weeks Cultures

MacConkey's (MAC)

MacConkey’s is both a selective & differential media.

1. What does it mean that MacConkey’s is selective??

2. What does it mean that MacConkey’s is differential?

Members of the family Enterobacteriaceae (Gram negative bacilli) are the most frequently encountered bacteria lactose fermenter isolated from many types of clinical specimens.

Blood agar (BAP)

Most specimens received in a clinical microbiology lab plated onto Blood Agar (abbreviated BAP).

Blood agar is usually inoculated from a patient’s throat swab.

Is this medium selective?

Is this medium differential?

What are the possible hemolysis patterns?

Microbiologist are trying to detect presence of Group A beta hemolytic Streptococci (Streptococcus pyogenes).

Other species can also cause B-hemolysis (Gram negative Enteric bacteria…poopie bacteria).

Mannitol Salt (MSA)

Is Mannitol Salt selective? Why?

Is Mannitol Salt differential? Why?

Recording Results from Last LabCultures

1. Describe results from touch plates and arm plates (TSY).a. What is the difference between plates 1, 2 and 3?

2. Record results of environmental samples, for each quadrants 1 – 4 (TSY and MacConkeys).

a. Are MacConkey colonies pink or colorless?b. What does it mean if they are pink?

3. Record results from throat swab plate (Blood agar).a. Do you see Alpha hemolysis? Beta hemolysis? Gamma hemolysis?

b. Which hemolysis pattern is bad news?

4. Record results from nasal swab plates (TSY and Mannitol Salt).a. Did any of the colonies turn the Mannitol Salt medium yellow?

b. What would it mean if they did?

II. Inoculation Loop & Streak Plate Method

We will culture our unknown onto MacConkey’s and Mannitol Salt.

To do a streak plate technique, we will need to use an innoculation loop.

• AKA smear loop, inoculation wand or microstreaker

• Simple tool used to retrieve an inoculum from a culture of microorganisms.

• Always sterilize in a flame until it becomes red hot before and after each use.

• By doing this, the same tool can be reused in different experiments without fear of cross-contamination.

• Be sure that your inoculation loop has cooled before using it to retrieve an inoculum or streak a plate!

• If you hear the medium sizzle… the loop’s too hot!

Streak Plate Technique

When we want to isolate a specific species of bacteria, we need to use a technique that will spread out the original “parent bacteria” in a sparse enough pattern so that after growth, individual colonies will result.

After incubation, the 4th quadrant of your plate should have dots. These small “dots” are individual colonies, and represent millions of bacteria of the same species

III. Differential Stains

Differential Stains

Most stains used in microbiology are differential stains.

Use more than one dye so that different cells,

chemicals or structures can be

distinguished.

Preparing Specimens for Viewing – Differential Stains

Gram StainDistinguishes between two large groups of microorganisms:

- purple staining, Gram-positive cells- pink staining, Gram-negative cells

These two types of cells differ significantly in the chemical and physical structure of their cell wall.

The structure of the thinner cell walls of Gram negative bacteria cannot hold the dyes previously used, once the decolorizer is applied.

Crystal violet (1 min) : rinse : Iodine (1 min) : rinse : Acetone Alcohol (10–15 sec) : rinse : Safrinin (1 min)

Mordant is a chemical used to fix the staining reaction

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Preparing Specimens for Viewing – Differential Stains

Acid-Fast StainThis stains the cells of the genera

Mycobacterium and Nocardia, which cause many diseases in humans, including tuberculosis, leprosy, and other lung and skin infections.

Cells of these bacteria have large amounts of waxy lipid in their cells walls, so the Gram stain and other water-based stains don’t work well on them.

Cells are determined to be acid-fast or not, based on whether they retain their primary stain after decolorization.

Create a smear of the organism that you are testing. Cover the smear with a strip of blotting paper.

Saturate paper with Ziehl’s carbolfuchsin and heat for 3 – 5 minutes. Remove blotting paper.

Wash slide with tap water and then decolorize the smear for 10 - 15 seconds with acid alcohol. Rinse again with tap water.

Apply crystal violet for 1 minute, wash, blot dry.

OBSERVATION OF MICROORGANISMS

Blotting paper : Ziehls carbolfuchsin (3 – 5 min heat) : rinse: Acid Alcohol (10 – 15 sec) : rinse : crystal violet (1 min)

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Preparing Specimens for Viewing – Differential Stains

Endospore StainSome bacteria produce endospores, dormant, highly-resistant

cells inside the cytoplasm of the bacteria, that can survive environmental extremes (desiccation, heat, harmful chemicals)

Most notable genera: Bacillus and Clostridium

Endospores cannot be stained by normal staining procedures because their walls are practically impermeable to all chemicals.

Endospore stain uses heat to drive the primary stain, malachite green into the endospore.

After cooling, the sample is decolorized with water and counter stained with safranin.

Results in green stained endospores and red-colored vegetative cells.

Malachite Green (5 min heat) : rinse : safrinin (20 sec)

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