Microbiology LAB Exam #1

Microscope- The Condenser

focuses the light ging into the objective lens

Microscope- The Iris Diaphragm

adjusts the amount of light going into the objective lens

Microscope- Nosepiece

holds the objective lenses and rotates to change from one objective to another

Microscope- Coarse Objective Knob

moves the stage larger distances to help get the object in focus

Microscope- Fine Adjustment Knob

moves the stage smaler increments to help get the object in better focus

Microscope- Objective Lens

magnifies the object. It is located near the object.

Microscope- Occular Lens

magnifies the image of the object from the objective lens. It is located near the eye.

Steps in using the microscope

1) scanner power in place

2) place slide of specimen stage in place with stage clips

3) Using the course adjustment knob, raise the stage all the way up and then down slowly until you can see image of specimen

4) Readjust with fine adjustment knob

Describe the purpose of the oil immersion lens

Used when oil is added to the slide. Oil must touch the object and objective lens to that the light does not refract futher

Parfocal

Parfocal microscopes are able to move from one objective to the next and still be in focus due to the length of the objective lenses

Resolution

The ability to distinguish between two objects that are very close together.

What is resolving power?

The measurement of resolution

What are two ways to increase resolving power?

1. By decreasing the wavelength of light (using a blue filter) 2. Increasing the numerical aperature

What units are used to express resolving power?

nm or nanometers

What is better, smaller or larger resolving power?

Smaller is better

Light microscopes have a 200nm resolving power

Electron microscopes have a .2nm resolving power

What is chromatic aberration?

The effect when many colors are seen toward the edge of the microscope field of vision (like a prisim)

How are microscopes corrected for chromatic aberration?

With a special achromatic lens (corrected for red and blue light) or by using a blue filter. The lab has blue filters in their microscopes which also increase resolution.

Describe the process of making a wet-mount slide

1) Add the specimen to the slide

2) Touch the edge of the cover slip with one edge of drop of specimen.

3) Let the cover slip drop gradually to the slide, covering the drop of specimen.

Describe the purpose of making a wet-mount slide

To be able to see living organisms under the microscope in their natural state.

What is the advantage of using a wet-mount slide?

Can see movement and determine if microbes posses their own method of motility. Can also see the actual shape and arrangement of microbes.

What is the dis-advantage of using a wet-mount slide?

The movement is very fast, microbes are transparent. Must use a "dark-field stop".

What is a producer?

Are autotroph organisms that make their own food (through photosynthesis).

What structure is an indicator of a producer?

Chloroplasts is an indicator that an organism is a producer.

An example of a producer is what?

Algae are producers that are found in a pond.

What is a consumer?

They are heterotrophic organisms that get their food from another source (nonphotosynthetic)

Examples of a consumer are what?

Protozoans such as paramecium and multicellular animals like rotifers or daphnia.

What is osmosis?

The diffusion of water

What organelle of protozoans responds to osmosis?

The cell membrane is where diffusion occurs.

Why do we use asephtic technique of bacteria?

To obtain pure cultures and to prevent contamination of pure cultures.

When is it best to use a broth?

When you want to grow a lot of bacteria.

When is it best to use a agar slant?

To subculture bacteria that does not need to be "isolated" and to store the bacteria for several months.

When is it best to use an agar deep?

Used to determine motility and oxygen requirements of bacteria.

Describe the process of aseptic transfer from broth to agar slant

1) Flame your loop

2) Hold broth at 45 degrees and pull off cap

3) flame lip of tube

4) obtain loopful of culture

5) flame lip of tube and replace cap

6)pull cap flame lip of slant

7)insert loop and zig-zag up the butt of the slant

8) flame lip of slant and insert cap

Describe the process of aseptic transfer from broth to agar plate

1) Flame your loop

2) Hold broth at 45 degrees and pull off cap

3) flame lip of tube

4) obtain loopful of culture

5) flame lip of tube and replace cap

6) open plate and zig-zag loop (without digging) across the surface of the agar

7) replace lid to plate

Why is the top of the tube heated?

To force out air and keep contiminants from settling inside.

When is a needle used rather that a loop when transferring bacteria?

1) To pickup specific colonies without accidently touching other colonies of other species nearby.

2) Also used to break up large chucks of bacteria

3) Used to inoculate agar deeps

define and identify the following broth culture growth patterns -Flocculant

clumps of growth throughout

define and identify the following broth culture growth patterns -Pellicle

A film of growth at the top of the broth

define and identify the following broth culture growth patterns -Sediment

Precipitate that falls to the bottom of the tube

define and identify the following broth culture growth patterns -Turbidity-

Growth evenly distributed throughout.

define and identify the following agar slant growth patterns -Arborescent-

branched growth from the original streak

define and identify the following agar slant growth patterns -Beaded

Isolated colonies toward the top of the slant

define and identify the following agar slant growth patterns -Echinulate

pointed (spiny) edge of growth

define and identify the following agar slant growth patterns -Filiform

Evenly distributed on slant (only where streaked).

define and identify the following agar slant growth patterns -Rhizoid

Root like distribution of growth (larger branches coming from the streak).

define and identify the following agar slant growth patterns -Spreading

Growth has spread out beyond the original streak

Describe the purpose of staining

To add contrast between the background and the microbe.

What are the advantages of using a simple stain?

Quick and easy to perform. Can see the shape and arrangement of the cells.

What are the disadvantages of using a simple stain?

Cannot differentiate the Gram Positive and Gram Negative cell wall based on this .

Differentiate between acidic and basic dyes

ic dyes have their color pigment on the negative ion and therefore are repelled by the cell, but stain the background. Examples: Congo Red, Nigrosin, and India Ink.

Basic dyes have their color pigment on the positive ion and therefore are attracted to the cell and stain the cell. Examples: Methylene Blue, Crystal Violet, Safranin, Malachite Green, and Carbol Fuchsin. There are many more examples - most dyes are basic.

Which dye stains the cell?

Basic dyes stain the cell

Which type of dye stains the background?

Acidic dyes stain the background.

What is the purpose of "fixing" the bacteria?

To attach the bacteria to the slide (so they won't wash away with the rinse.)

What are two different methods for fixing the bacteria?

Heat fixing is what we do - running the slide through the Bunsen burner for a couple of seconds. Chemical fixing uses methyl alcohol to fix the bacteria. In addition to making it stay on the slide, it kills the cells.

Describe the purpose of the gram stain.

To distinguish between cells that have a Gram Negative and Gram Positive Cell Wall.

Describe the process of the gram stain

o Smear bacteria onto slide as normal. Let dry.

o Heat fix. Let cool.

o Crystal violet - primary dye - for 1 minute. Rinse with water.

o Iodine - mordant - for 1 minute. Rinse with water.

o Ethanol - decolorizer - for 30 seconds. Rinse with water (to stop decolorizing).

o Gram's Safranin - Secondary dye (counterstain) - 1-5 minutes (depending on cultures and strength of dye.)

List the primary dye, counterstain, and decolorizer used in this stain

o Primary - Crystal Violet

o Counterstain - Safranin

o Decolorizer - Ethanol 95%

Discuss the purpose of a mordant and list the mordant used in the Gram Stain

Mordant chemically changes dye so that it is not able to leave the cell wall. It is trapped in the peptidoglycan layer.

Describe differences in staining as they pertain to the differences in the structure of the cell wall.

o Gram negative - has an outer membrane of phospholipid that surrounds the thin peptidoglycan layer. It is able to be dissolved with alcohol and therefore the dye is removed during the decolorizing step with alcohol.

o Gram positive - has a thick peptidoglycan layer and no outer membrane of phospholipids. Therefore, it is not decolorized by alcohol.

Identify cells as Gram positive or Gram negative according to the color of their gram stain

o Gram positive - purple

o Gram negative - red/pink.

Explain why alcohol washing step is critical to the Gram staining process.

If no alcohol is applied, then all the cells will remain purple and no distinction between Gram + and - will be seen.

Describe the purpose of the Acid-Fast Stain

o Purpose - to distinguish between cells that are acid-fast and those that are non-acid fast. To determine if bacteria in sputum is acid fast, indicating that it might be Mycobacterium tuberculosis

Describe the process of the Acid-Fast Stain

o Smear bacteria on slide as normal (however, with Mycobacterium it is usually clumpy and will require a needle to separate the clumps in the smear.) Air dry.

o Heat Fix. Let cool.

o Apply Carbol Fuchsin dye to bacteria for 15 minutes over boiling water to steam the dye into the bacteria. Rinse with water.

o Decolorize with Acid Alcohol for 30 seconds. Rinse with water.

o Apply counter stain - Methylene blue for 1 minute. Rinse.

Identify the following: primary dye, counterstain, and decolorizing agent.

o Primary Dye - Carbol Fuchsin

o Counterstain - Methylene Blue

o Decolorizer - Acid Alcohol (3% Hydrochloric Acid + 95% Alcohol)

Explain why cells that are acid fast appear red and non-acid fast appears blue.

o Acid fast cells will be stained by the carbol fuchsin and will appear red because they do not decolorize easily (not even with acid alcohol.)

o Non-Acid Fast cells - will decolorize with acid alcohol and when counterstained will appear blue (from the Methylene blue dye.)

o If only alcohol was used for the decolorizer, some non-acid fast cells would not be decolorized (Gram + cells that were non-acid fast would retain the dye because the 95% alcohol is not strong enough to decolorize them. To decolorize non-acid fast-gram positive cells, you must use acid alcohol.)

Describe differences in the cell wall of acid-fast and non-acid-fast cells

o Acid fast cell walls have a waxy cell wall - due to the Mycolic acid in their cell wall

o Non-acid fast cells do not have the waxy cell wall - no Mycolic acid.

Give examples of disease-causing acid-fast bacteria

o Tuberculosis and Leprosy are caused by bacteria in the Mycobacterium genus.

Capsule stain

Describe the purpose of a capsule stain

to view the capsule or determine if a capsule is present

Describe the process of the capsule stain

o Smear as usual.

o No Heat Fixing. Let it air dry.

o Apply an acidic dye (like Nigrosin or India ink) in a thin smear - let air dry. Remove excess dye. NO RINSE.

o Apply a basic dye on the stained slide for 1 minute. Remove excess dye. NO RINSE.

o Blot dry. View

List two functions of a capsule

o Anti-phagocytic factor - makes it difficult for a phagocyte to engulf and "digest" it.

o Keeps the bacteria from drying out.

Identify the name and type of dye used to stain the cells in a capsule stain

Basic dyes, like Maneval's, stain the cell of the bacteria.

Identify the name and type of dye used to stain the background in a capsule stain

Acidic dyes stain the background around the bacteria

Describe the appearance of bacteria and capsules when using this stain technique

Bacteria are surrounded by a halo of white (the capsule) with a dark background

What is the purpose of this stain?

To view the spore or determine if the species of bacteria produce a spore.

What is a spore?

A thick layer of polysaccharide that encloses the genetic information and enzymes needed to maintain life at minimal metabolic levels until conditions are right for normal metabolism, growth and reproduction. It is a survival mechanism, not a reproductive structure. One spore produces one cell.

What is the primary dye for spores?(name and what does it stain)

Malachite Green - it stains the spore

What is the counter stain for spores? (name and what does it stain)

Safranin - it stains the vegetative cell around the spore.

What is the decolorizing agent for spores?

Water

What are examples of disease causing genera and the diseases they cause.?

o Clostridium and Bacillus

o Tetanus, Botulism, Anthrax, food poisoning

What is an autoclave?

An autoclave sterilizes media and instruments (inanimate objects) by using pressurized steam.

How is heat generated in the autoclave?

Water in the container is heated in a sealed compartment. The steam has no where to expand to and therefore pressure builds up, allowing the steam to reach 121˚C.

At what temperature, pressure, and time was required to sterilize our TSA medium using the autoclave?

The autoclave will reach 121˚C at 15 pounds of pressure. 15 minutes is required to sterilize a normal load (however if larger amounts of liquid are being sterilized, more time may be required.)

Explain the use of the ampules (Bacillus stearothermophilis endospores) in autoclaving.

The Bacillus stearothermophilis is a spore-forming thermophile. Therefore it is difficult to kill with boiling and the temperature must reach 120 or more to kill this bacteria. If this bacteria is killed, then all other non-spore-formers that are not thermophiles, should be killed as well. The ampoule contains bromthymol puple - pH indicator. If fermentation of the bacteria occurs (resulting from germination of the spore), it will produce an acidic environment and will change the pH indicator to yellow. This visible result will clearly indicate that fermentation has occurred and that sterilization was not achieved

Define the term sterilization

Absence of all microorganisms (spore-forming and viruses). Mcrobes are no longer able to reproduce

What are the dissolving and solidifying temperatures of agar?

The dissolving temperature is between 97-100 ˚C. The solidifying temperature is 42 ˚C.

Describe the purpose of the streak plate and pour plate

Streak plates allow isolation of colonies of a sample of bacteria on the surface of an agar plate from undiluted samples. The bacteria are diluted from one section of the plate to the next by flaming the loop and only bringing a small portion of bacteria to the next section of the plate (by streaking).

Spread plates allow isolation of colonies of a sample of bacteria on the surface of an agar plate from diluted samples.

Pour plates allow isolation by dilution of bacteria to levels that allow isolated colonies throughout agar.

Compare and contrast these three techniques by pattern of growth on the surface

Streak plates require dilution by flaming the loop after streaking each quadrant and only taking 3 streaks from the previous quadrant into the new quadrant. By doing this, each quadrant is more dilute than the previous quadrant. The 4th quadrant should always be dilute enough to contain isolated colonies.

Pour plates have isolated colonies of bacteria evenly spread throughout the medium (not just on the surface, but all throughout the agar.). In the Pour Plate procedure, the bacteria is diluted in sterile "water blanks" prior to plating the bacteria and pouring agar over the bacteria. The bacteria sample that is dilute enough will produce isolated colonies throughout the medium (rather than just on the surface.)

Spread Plate procedure is similar, to the pour plate in that the bacteria is diluted prior to inoculation on the plate. The bacteria sample is placed on an agar plate and then spread evenly across the surface of the agar (rather than throughout the medium).

Compare and contrast these three techniques by where bacteria grow in the media

Streak Plate - bacteria grow only on the surface

Pour Plate - bacteria grow throughout the media

Spread Plate - bacteria grow only on the surface

Differentiate between a pure culture, mixed culture, and contaminated culture.

Pure - only one type of bacteria is present in a culture.

Mixed - two or more types of bacteria are contained within a culture (intentionally.)

Contaminated - two or more types of bacteria are found in a culture (unintentionally.)

Explain the purpose of inverting plates when incubating.

Inverting plates makes sure that condensed water does not fall on the surface of agar plates. Water that falls on the plate will mix different colonies together and not allow isolated colonies to appear on the surface

What is the purpose of the Standard Plate Count?

To determine how many Colony Forming Units/ml are present.

What is the range CFUs/plate that are countable?

30-300 CFUs per plate are statistically significant.

What are two assumptions that are made in the SPC?

1) That all bacteria will grow in the media and under conditions provided.

2) That one bacteria results in one colony forming unit.

Describe how to make a specific dilution (like 1:10 or 1:100).

To make a 1:10 dilution, 1 ml of the sample is added to 9 ml of sterile water. This gives a 1:10 dilution because the dilution is calculated by amount added divided by the final volume. (1/1+9 = 1/10)

Describe how to calculate the dilution, total dilution, and number of CFU's/ml of the original sample.

Calculate dilution by taking the amount added divided by the final volume (ie. If you added 1 ml to 9 ml, then 1/1+9= 1/10 (1 ml added, 10 ml was the final volume).

Calculate total dilution by multiplying the dilutions - for example, if we diluted a sample 4 times - 1ml added to 9ml each time - then the dilution is a 1:10 of a 1:10 of a 1:10 of a 1:10. In mathematical sentences, "of" means multiply. So, 1/10 x 1/10 x 1/10 x 1/10 = 1/10,000. (notice that there are 4 dilutions of 1/10 and the final dilution has 4 zeros. Count the zeros in your problem and in your answer to check yourself.)

CFU's/ml - The CFU's are colony forming units. Since we assume that one colony is the result of 1 bacteria cell which originally was inoculated on the plate (not two or more that were very close together), we call them colony forming units.

The formula is: CFUs/ml = Total CFUs on the whole plate / amount plated x dilution 1. Determine the amount of CFUs on the whole plate. Many questions will give the amount on a 1/4th or ½ of the plate. That is not the whole plate. To estimate the amount on the whole, multiply by 4 (if 1/4th is given) or 2 (if ½ is given).

2. Determine the total dilution.

One thing to remember - if you plate 0.1ml instead of a full 1.0ml, your total dilution on the plate is 1/10 of your dilution. Unless otherwise stated, assume that 1ml of your diluted sample, was plated.

Multiply the total CFUs/plate x the inverse of the total dilution. (The dilution is a fraction. When dividing by a fraction, you are essentially multiplying by the inverse of the fraction. Therefore, CFUs x inverse of dilution is another way to look at it.)

Convert Fahrenheit to Celsius

C = 5/9 (F - 32)

Convert Celsius to Fahrenheit

F = (9xC)/5 +32

= How many decimel places, to the right of base

Six places --- .000 000

nm= How many decimel places, to the right of base

Nine places --- .000 000 000

6.02x 10 = moving the decimel over how many spaces?

23