BIOL 351 - Microbiology Lab Exam 1

Ubiquitous Nature of Organism

Can come from basically anywhere including soil, water, plants, animals and even from hot acid pools and uninhabitable sites

Free living microorganisms

Do not reside on or in a specific plant or animal host

Nonpathogenic

Not known to cause disease

Symbiotic microorganisms

Organisms that reside on or in a host

Symbiotic examples AND definition ( 3 examples)

• Pathogens (cause damage to host)

• Mutualism (both host and microbe benefit)

• Commensals (microbe benefits but no effect on host)

Opportunistic Pathogens

Inhabit our bodies and are capable of producing a disease state if introduced into a suitable part of the body

Reservoir

Any area where a microbe with the potential to cause infection resides

Saprophytes

Organisms that decompose organic matter

Purpose of blood agar

Differentiate bacteria based on their hemolytic characteristics

Blood agar composition

Includes 5% sheep blood in a Tryptic Soy Agar base

Hemolysins

exotoxin in gram (+) that can destroy RBC/s and hemoglobin

β – hemolysis

Complete destruction; results in clearing of the medium

α – hemolysis

Partial destruction; results in greenish discoloration of the medium

γ – hemolysis

no hemolysis

β – hemolysis IMAGE

α – hemolysis IMAGE

γ – hemolysis IMAGE

Germicides AND what are three categories

Substances or systems, both chemical and physical, that prevent the spread of pathogens:

Decontamination

Disinfection

Sterilization 

Decontamination

Reduction of pathogenic microorganisms to a level at which items are safe to handle without protective attire

Lowest level of control

EX: physical cleaning with soaps or detergents

Disinfection

Kill most, if not all, of the targeted pathogens but typically do not kill large numbers of spore

Next level of control

Typically liquid - can include dry heat, moist heat, ultraviolet light

EX:

• Chemical sterilant: 

  • High-level disinfectants that have the ability to kill all vegetative cell and some spores

• Antiseptics:

  • Disinfectants designed to reduce or eliminate pathogens on or in living tissue 

Sterilization

Complete elimination of viable organisms including spores

Highest level of pathogen control

Through chemicals, gases, incineration, oxide gas, low temp plasma, etc

Steam sterilization

Most effective and most common method

Temp: 121-127 C for 15 minutes

EX: autoclave

• Superheated steam under pressure to kill heat-resistant organisms

Steam sterilization - Biological indicators

only way, with certainty, to determine that sterilization has been achieved.

Indicator vial includes:

• Small ampule containing fermentation broth with pH indicator

• Strip of filter paper containing bacterial spores

Test:

• Vial is autoclaved at 121°C for 15 minutes

• Ampule is crushed

  • Allows fermentation broth to come into contact with bacterial spores

• Vials are incubated at 55°C for 48 hours

Since microorganisms are ubiquitous, do you expect growth on all the plates?

No, this is because the environment must be perfect for the microorganism, so even though it could be exposed to contaminants, growth may not be present if environment is not ideal.

Would growth occur on control plate?

No, this is because it was not exposed to any contaminants and was in a sterile environment for the experiment.

Would you expect the same number and type of organisms to be obtained from different environments?

No, this is because the conditions for each microorganism to grow vary. Therefore, specific environments would inhibit or facilities growth in certain organisms.

Why is morphological characterization useful?

first indication one organism is different from another

What exotoxins do streptococci produce and what do they do?

streptolysins -- damage RBC

Different bacterial colony shapes

round, circular, irregular, punctiform, filamentous, rhizoid

punctiform

tiny, pinpoint

filamentous colony

long strands of bacteria growing end to end, resembling hair strands

rhizoid colony

thin, branching projections

types of margins

smooth, lobate, undulate, filamentous, rhizoid

lobate margin

marked indentations

undulate margin

wavy

filamentous margin

threadlike, spreading edge

types of elevations

flat, raised, convex, umbonate, crateriform

umbonate elevation

raised in the center

crateriform

cup-shaped

convex elevation

dome-shaped elevation

what is aseptic technique?

A procedure performed under sterile conditions, going from pure culture to pure medium

Small Slant or a Stab

used when you want to grow an organism in anaerobic conditions

Streaking slanted portion of media (aerobic)

A liquid culture

When you want to increase the number of cells of a particular microorganism

• exponential growth

A slant

Simultaneously grow an orgnaism in anaerobic conditions (stab media) and also aerobic conditions (streak across top of slanted media)

• advantage: better contains liquids bc in tube

Agar plate

Take mixed culture and spread it thin “streaking the plate” so individual bacteria is separated from one another

USED TO ISOLATE SPECIES

Aseptic Transfer

Transfer living microbes from one place to another without contamination of:

• the culture

• the sterile medium

• or the surroundings

A culture

any medium that contains living microorganisms

• pure culture: one organism

• mixed culture: multiple organisms

Tips for maintaining a pure culture

1. clean hands and surface

2. take time and be organized

3. Have test tube rack

4. Use incinerator on loop before and after transferring culture

5. Do not talk or be distracted

How to hold inoculating tube?

I. Hold like pencil in dominant hand at a slant and hold cap in dominant pinkie

II. Lid is not taken off plate

What NOT to do when holding inoculating tube?

I. Do NOT take lid off agar plate and place on counter

II. Do NOT forget to slant tube —> prevent dust from contaminating

Writing Organism Names

While typing

1. Escherichia coli

2. E. coli

While writing

1. Escherichia coli

2. E. coli

To prevent contamination - inoculation

• wire loops are sterilized immediately before use in incinerator

• cool loop in agar before transfer

• mouths of tube with culture is incinerated

• instruments that can not be incinerated are sterilized inside wrappers or containers by autoclaving

Broth Use

grow microbes when fresh culture or large number of cells are required

USE TO GROW CULTURES

Agar slants

grow stock cultures that can be refrigerated after incubation and maintained for several weeks

WHEN YOU WANT TO GROW A CULTURE TO STORE IN THE FRIDGE

Plated media

obtaining isolations of species differential testing and quantifying bacterial densities

Methods of Isolation

bacterial sample assumed to be mixed culture - ALWAYS

• streak plate

• pour plate

• spread plate

Purpose of streaking for isolation

Obtain isolated colonies —> composed of a million identical cells

• can obtian pure culture from this —> single colony

Quadrant streak method

bacterial sample streaked over surface of agar medium

• cell density decreases —> individual cells deposited separately on agar surface

• results in colony only of original cell type

• used for samples with high cell density

Properly streaked plate

Not sterilizing loop between quadrants

Improperly streaked quadrants

result of not rotating plate properly

Loop was not cooled or loop did not pass through previous quadrant

Growth Characteristics are Influenced By:

Nutrient Availability

Temperature

Incubation Time

Categories of Texture

mosit, mucoid, butyrous, dry, shiny, dull

Organisms cultured on slat - growth characteristics

filiform, friable, spreading edge, pigmented or translucent

Filiform

dense and opaque with a smooth edge

Friable

crusty

Spreading edge

produced by motile organisms

Microorganisms culture in a broth - growth characteristics

pellicle, sediment, uniform fine turbidity, flocculent

Pellicle

growth floats on top of medium

Sediment

Growth sinks to the bottom

Flocculent

clumped growth

Where would you expect to find a saprophyte?

In soil

What would result in pure culture?

A completely isolated colony growing on a plate

Microorganisms are ubiquitous. This means they would be found in which of the following places?

How hot can your inoculating loop get if you leave it in the incinerator for a really long time?

close to 800 degrees

How would you identify the only lens in which you would use immersion oil?

It has a black and white ring around it

Which of the following is the reason a simple stain will stick to a bacteria?

 

They are positively charged and the bacteria are negatively charged

How much water would you use on a slide when doing a bacterial smear procedure?

 

A very small amount

What is the correct term for a chemical that helps to fix another chemical in place by complexing with it?

 

mordant

After which of the following steps in the Gram stain process could you detect a difference between Gram + and Gram- bacteria if you looked really hard?

 

After de-colorization with alcohol

Which of the following is NOT a source of getting a poor/ inconsistent Gram stain?

 

Making sure to air dry the smear before heat fixing

Which of the following explanations is correct as to how a negative stain works?

 

They are negatively charged and therefore are repelled by the bacteria, and surround it so that it stands out.

Under which circumstances would you choose a negative stain protocol over a Gram stain?

 

If the bacteria would lose their delicate shape or shrink when heat fixing

Micropipette

Used for transfer of small amount of liquid

Micropipette tip color

Clear: P1000

Yellow: P20 P200

P1000 Range IMAGE

P200 Range IMAGE

P20 Range IMAGE

Pour Plate Technique

Yields isolated colonies of bacteria and fungi

Spread Plate Technique

method of isolation in which diluted microbial sample is deposited on agar plate

SERIAL DILUTION CALCULATIONS

yes

Know Microscope Anatomy

yes!

Image Formation

1. light source

2. light pass through condenser

3. light pass through objective lens —> refracted —> real image

4. Image is magnified —> virtual image

Brightfield IMAGE

Darkfield IMAGE

Phase-contrast IMAGE

Flourescence IMAGE

Darkfield microscopy

light travels in a path past the objective unless scattered into it