Micro lab practical

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How do you total the magnification of a microscope?

x by 100

Characteristics of Protozoa?

-Has a nucleus (some have 2 nuclei)
- Unicellular
- Lack a cell wall
- Reproduces asexually(most) or sexually
- Mitosis(most) or Meiosis

What methods of motility does Protozoa use?

- Cilia
- Flagella
- Pseudopod (except subgroup Apicomplexa)

What kind of enviroment does Protozoa like?

Moist enviroments
EX: Ponds, streams, lakes, and oceans.
Some like moist soil, beach sand, and decaying organic matter.

Are protozoa's usually pathogenic?

Very few are pathogenic.

Are most protozoa chemoheterotrophic?

Yes

How doe protozoa obtain their nutrients?

By phagocytizing bacteria, decaying organic matter, other protozoa, or tissue of their host.

Why is agar a good choice to use for growing cultures?

provide a solid medium on which microbes may be cultured. They remain solid, as very few bacteria are able to decompose agar. It melts at a higher temp.

What controls the amount of light reaching the ocular lens?

The iris diaphram

What is TSA?

It is a general purpose medium , providing enough nutrients to allow for a wide variety of microorganisms to grow. It is used for a wide range of applications including; culture storage, enumeration (counting), isolation of pure cultures or simply general culture. e.g. Tryptocase Soy Agar.
**Incubate for 24hrs at 37 degrees celcius**

What is TSB?

Trptocase Soy Broth

Innoculation

Introduction of a sample of microorganisms into a container of media to produce a culture of observable growth

Turbidity

The measurement of water cloudiness; it may be affected by such things as sediment and plankton concentrations

Colony

In microbiology, a group of organisms grown from a single parent cell.

Colony forming unit

A unit of measure for microbes, a population of cells that arises from a single bacterial cell.

What is the appearance, margin, and elevation of bacterial colonies?

1-3mm in diameter, circular appearance, entire margin, and flat elevation.

What is the appearance, margin, and elevation of fungal colonies?

Circular and sometimes hairy like appearance, Entire margin, rounded elevation.

Flocculent

Clumps of growth in a medium.

Sediment

The solid material that settles to the bottom of a liquid.

Pellicle

A firm, flexible coating outside the plasma membrane.

What are the aseptic transfer of and smear preperation of a specimen from a solid and a liquid media?

Solid- swab, dip in broth, seal in tube.
Broth- heat innoculating loop, briefly cool, dip in broth, then transfer to smear plate, heat loop again and let cool.

What is the purpose of heat fixing?

It helps the cells adhere to the slide so that they can be stained.
The purpose of heat fixing is to kill the organisms without serious distortion. They adhere better to the slide and also take up dye more easily.

Simple stain

It involves the use of a single stain which enables the microorganism to be more readily observed.

Differential stain

It uses more than one stain to differentiate bacteria or structures (Gram stain, Ziehl Nielsen stain, etc)

Structural stain

It is used to identify cell parts not revealed by conventional staining (spores,feulge,flagellar,capsule).

Why do basic stains adhere to bacterial cells?

Basic stains typically have a colored cation such as crystal violet combined with a colorless anion such as chloride. Bacterial cell walls have a negative charge so the positively charged crystal violet binds to the negatively charged cell wall.

What are the basic morphologies and arrangements of bacterial cells?

Morphology-The basic forms are spheres (coccus) and round-ended cylinders (bacillus). But there may be others such as helically twisted cylinders (spirochetes).
Arrangement- They also conform diplos(2), tetrads, staphylos(Grapelike cluster), streptos(chain), palizadas(stacked).

What are the steps of the gram staining process?

1) Flood with crystal-violet dye (heat fixed smear)
2) Flood with iodine- which binds to crystal violet and traps it in the cell.
3) Use ethanol & acetone- Rapid decolorization
4) Use safranin to counterstain

What color are gram + cells and why?

Purple- Has a thick cell wall that retains the crystal-violet.
*Step 1-4 gram + cells stay purple.*

What color are gram - cells and why?

Pink- Has a thin layer of peptidoglycan that does NOT retain crystal-violet.
**Gram - cells are purple step 1-2, colorless in step 3, and pink in step 4.

What are some reasons a gram+ cell would stain -?

Age of the culture. Cultures should be 18 to 24 hours when used for a gram stain.
2. Decolorizing too long. It is important to have a known gram positive organism on the slide as a control. If the control smear is not purple then the results are unreliable.
3. Gram's iodine may deteriorate over time. If the iodine solution is weak, fixing of the crystal violet may not occur allowing the crystal violet to be removed too easily.
4. Over heating of the smear can damage the cell walls allowing decolorization

What are the + or - results of staphylococcus epidermis, E. coli, and Bacillus substilis?

Staphylococcus epidermis = Gram + (purple), Coccus shape, and Staphy arrangement.
E. coli = Gram - (red/ pink), Bacilli shape, and Palisades arrangement.
Bacillus subtilis = Gram -, bacilli(rod) shape, Strept(chain) arrangement.

Acid-fast staining

•Important diagnostic stain
•Differentiates acid-fast bacteria (pink or red) from non-acid-fast bacteria (blue)
•Important in medical microbiology
•Mycobacterium

Why do some bacteria stain red in acid fast staining?

-Acid-Fast bacteria (AFB) are organisms covered with a hard waxy outer layer called lipoid capsule.
-The high mycolic acid content of certain bacterial cell walls, like those of Mycobacteria, is responsible for the staining pattern of poor absorption followed by high retention.

What are the reagents of the acid-fast stain?

A prolonged application of primary stain to a smear; washing the stained smear with methanol and hydrochloric acid (decolorizer); and application of counter stain to the washed smear, which identifies the acid-fast bacteria from the rest of the organisms found in the smear.

What are the regeants of the capsule stain?

In this procedure violet crystal is always used first, a copper sulfate solution will then be applied to serve as a counterstain. After this is done, the violet will be removed from the cell and it will appear as a whitish-blue halo.

What is the appearnce of the positive capsule stain?

Looks like a halo; colorless around a pink cell.

Spore staining?

Endospores will be green inside a pink cell.
If they are vegetative, the green dye will be washed out with the de-ionized water. It will then be clear. The counter stain will then dye the cell pink, but leave the endospore green.

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