Microbiology Lab 1

Microbial Sampling (what does the n represent)

Sample Size

What are common issues that arise in microbial sampling?

1) Contamination
2) Temperature Fluctuation
3) Sampling Frequency

Why is temperature fluctuation an issue in sampling?

refrigeration for too long allows competing bacteria to grow; freezing can kill pathogens

What are the conditions one must consider for microbial growth?

Temp (20-37C), Light, Salinity, pH (6.5-7), Nutrients

What are the four phases of microbial growth?

Lag Phase, Log Phase, Stationary Phase, Death Phase

What does each phase mean (in order)?

1. No increase in the number of living bacterial cells

2. Exponential increase in the number of living bacterial cells

3. Rate of cell division and death are roughly equal

4. Exponential decrease in the number of living bacterial cells

What is this media?

Nutrient Agar: is the most commonly used, with organic nitrogen. 0.3% beef extract/yeast extract. Non-fastidious organisms.

What is this media?

Blood Agar is also a general nutrient broth, however it contains 5% sheep or horse blood. It is useful for cultivating fastidious organisms and for determining the hemolytic capabilities

What media is this?

Eosin methylene blue (EMB) is a selective stain for Gram-negative bacteria. A differential test uses special lab growth plates to separate harmless bacteria from fecal bacteria that might indicate dangerous germs in water.

Morphology

the study of form and structure

What are the different shapes? (Name 7)

1. Coccus (spherical)

2. Bacillus (Rod)

3. Spiral

4. Box-Shaped

5. Appendaged

6. Pleomorphic

Why do we do Microbial Isolation?

Decrease population density and isolate individual colony

How do we do serial dilution?

Broth or Direct Plating
9mL of broth
1 mL of Inoculum

What does a gram stain results mean?

Gram-positive: These bacteria retain the purple/blue dye because they have thick cell walls made of peptidoglycan
Gram-negative: These bacteria lose the purple dye and pick up a pink/red counterstain because they have thin cell walls with high lipid content

What type of stain is this?

Gram Stain

What does a simple stain show?

reveals basic bacterial cell shape, cell size, and cellular arrangement under a microscope

What stain is this?

Simple Stain

What are the steps for a gram stain?

1. Prepare a bacterial smear on a clean slide.

2. Air-dry, then heat-fix.

3. Add crystal violet → rinse with water.

4. Add Gram’s iodine → rinse with water.

5. Add decolorizer (75% ethanol) → immediately rinse with water.

6. Add safranin → rinse with water.

7. Blot dry and observe under the microscope.

What does India Ink reveal?

Reveals whether bacteria have a capsule

India ink is a ________ stain

negative: stains background rather than bacterial cells

What type of stain is this?

Methylene Blue Stain

What type of stain is this?

Acid-fast stain

What does an Acid-Fast stain detect?

Detects specific types of bacteria that have waxy, mycolic-acid-rich cell walls

What does an endospore stain reveal?

The presence of bacterial endospores

What is this stain?

Endospore Stain

dilution streak plate method

streaking from quadrant 1, from 1 to 2, from 2 to 3, and from 3 to 4

Macronutrients

elements microbes need in relatively large amounts to build cells

What are some macronutrients needed by bacteria?

carbon, nitrogen, phosphorus and sulfur

What are micronutrients?

needed in much smaller amounts

Examples of Micronutrients

iron, zinc, and manganese

What happens if the improper nutrients are available for bacteria?

The bacteria will have poor or no growth

What are the six features of colony observations?

1. Size

2. Color

3. Texture

4. Elevation

5. Form

6. Margin

Antibiotic

A substance that kills bacteria or slows their growth

Mueller-Hinton Agar

A standardized growth medium used to test how bacteria respond to antibiotics

Disk Diffusion Test

A bacterial lawn covers the plate. Paper disks containing antibiotics sit on the agar, and the antibiotics diffuse outward

Zone of Inhibition

Clear area around a disk where bacteria did not grow. Diameter is measured in millimeters

What are the classification used for zones of inhibition?

susceptible, intermediate, or resistant

Does the largest zone identify the best antibiotic?

No because each zone must be compared to a reference table of that bacterium and antibiotic classify the results

What are the four components of differential staining?

1. Primary Dye

2. Mordant

3. Decolorizing Agent

4. Counterstain

What stains are positive and negative?

Simple, Gram, endospore, and acid-fast stains use positive dyes. Negative staining colors the background.

Endospore uses ____ stains

two

How is the first stain in the Schaeffer-Fulton method stick to endospore

heat

What colors do you see for endospore staining?

The vegetative forms will take up the pink/red stain from safranin while the endospores will stain green, from the malachite green dye.

What colors do you see for Acid fast staining?

Red/Magenta - Acid Fast
Blue or Purple = Non Acid Fast

Durham Tube Protocol

A Durham tube is provided in tubed broth media to collect the gas produced during fermentation

What does an oxidase test determine?

Determines whether an organism posses the cytochrome C oxidase enzyme

What are the results for oxidase?

Purple: Oxidase-positive
Clear: Oxidase-negative

What does the Simmons citrate test determine?

determines whether bacteria can use citrate as their only carbon source to grow.

What are the color results of Citrate Test?

• Positive: growth, usually with the agar turning blue.

• Negative: no growth; agar stays green

In order to survive, organisms must rely on ________ __________ that allow them repair or ________ the ________damage of hydrogen peroxide (H2O2)

defense mechanisms; escape; oxidative

What enzyme facilitates in cellular detoxification? how?

Catalase by neutralizing the effects of hydrogen peroxide

What does the catalase test detect?

Detects catalase in bacteria

How do you interpret the results of the catalase test?

Bubble - catalase positive
No Bubbles - catalase negative

Microbial Identification

a process of hypothesis testing to provide evidence to support a proposed identification

Assays (general)

A standardized laboratory procedure used to detect, measure, or evaluate a specific biological or chemical property, activity, or response in a sample

Single Assays can provide a complete answer (T/F)

False

What does a plaque assay measure or detect?

Concentration of infectious bacteriophages (PFU/mL) on a susceptible host

What does Kirby-Bauer Disk Diffusion measure or detect?

Bacterial susceptibility to antibiotics based on zones of inhibition

What does PCR measure?

Presence of a specific DNA sequence

What does ELISA measure or detect? (2)

Presence of quantity of a specific antigen or antibody

What does a viable plate count (CFU) measure?

concentration of culturable microorganisms

What does a plaque assay measure or detect?

measure the concentration of infectious lytic viruses (viruses that cause cell death) by counting discrete, clear zones called plaques in a cell monolayer