Micro Lab Midterm

This will be a 27 question test(25 for a grade and 2 bonus questions for extra credit). Half of it will be question and answer on the paper, while the other half will be looking at slides or plates or showing procedures.

Topics:

Safety in the Lab:

PPE’s: lab coat, gloves, goggles, hair up, and no dangling jewelry, shoes with socks, no loose clothes…full pants on

Bags in locker, no food or drink in lab, use rubber bulb to pipette and not your mouth

No running, laughing, or reaching over flames

Know the difference between biohazard symbols and the radiation symbol

Know where the fire extinguisher is, and that it is a combination fire extinguisher equipped to handle all types of fires.

Know that we have a blanket in case you catch fire, and how to drop and roll to extinguish yourself.

If you get something splattered on your person, use the shower to dilute it and wash it off.

If something gets in your eye, use the eye flush fountain beside the shower.

Know that the first aid kit is by the cabinet, and that you are to notify in order to use something from there, so that the item can be replaced for quality control purposes.

Know where to throw out materials: Contaminated goes in the red bucket, non-contaminated in the regular trash can, and the broken glass containers are for broken glass and the sharps container are for razors, sharp metal, and needles

Flame all loops and needles to sterilize

Wash your hands and disinfect bench with ethanol before and after using the lab

Microscope Handling: Know how to carry the microscope—two hands…one on base and one on arm

1-3: Transferring liquid from a liquid microbe test tube to a test tube of nutrient broth.

Loosen both caps Turn on the flame and sterilize the loop Open the cap from the microbe and flame quickly the top of the test tube Introduce the loop by first cooling it off on the edge Grab a loopful of microbes Flame the top of the tube again and cap it shut Open the broth tube and flame the outside of it Put the loop into the broth and tap out any excess before taking it out Flame the tube before capping and labeling it Flame the loop so it can be used again

1-4: Know how to streak a plate in order to isolate colonies. Remember, you are to take the culture from the isolated colony in quadrant 4

Flame loop

Open cap and cool off loop, and obtain sample on loop, close cap on microbe

Remove cap on plate and drag loop from side to side and leaving a few zigzags towards the middle.

Flame loop, turn plate 45 degrees and cool off loop, do the zig zag procedure, making sure some won’t touch the sides.

Repeat two more times

2-1 Read the theory behind the ubiquity of Microorganisms, so that you understand the importance of washing your hands. She also said to make note of how mold can grow in air vents, and that if you were to culture them, they would look fuzzy, unlike bacteria.

Many microorganisms are free-living—they do not reside on or in a specific plant or animal host and are not known to cause disease. They are non-pathogenic.

Other microorganisms are pathogens—and are associated with hosts.

Many of the commensal or mutualistic strains inhabiting our bodies are opportunistic pathogens. They are capable of producing a disease state if introduced to a suitable or weakened part of the body.

Any place where a microbe resides and serves as a potential source of infection is called a reservoir.

2-2 Pay attention to the description of the shapes of cells in a colony. Look at the pictures in this section, especially the ones on the top of page 37 where they talk about features. Notice also the swarming grown pattern of P. vulgaris on p.40 (the concentric rings). Notice descriptions of pigments…for example M. luteus is light yellow, while S. aureus is gold. P. aeruginosa is green.

Shape may be described as round, irregular or punctiform

Margins may be entire(smooth), undulate(wavy), lobate(lobed), filamentous, or rhizoid(branchlike)

Texture: moist, mucoid, dry

Pigment production(color): opaque, translucent, shiny, dull

Elevation

also flat-raised margin, and raised spreading edge, and plateau

Margin

also irregular and rhizoid(branchlike)

(swarming)

Whole colony

also filamentous where there is skinnier and more numerous filaments

2-3 Growth patterns and pigment production on slants

Most organisms in this class produce **filiform growth on slants(dense and opaque with a smooth edge)…textures can range flat and dry for bacillus, to spreading edge for A. faecalis, crusty/friable(crunchy looking) for M. phlei, or transparent for lactobacillus

Colors can range from green(P. aeruginosa), white(Staphy epidermis), violet(chomobacterium), red(Serratia marescens), Rose(kocuria rosea), light yellow(m. luteus), gold(S. aureus)

2-4 Growth patterns in broth.

Uniform turbidity: even cloudiness(Enterobacter and Citrobacter)

Sediment: residue sinking down(Enterococcus and S. aureus)

Pellicle: membrane on top (Mycobacterium—waxy)

2-7 Fluid thioglycollate and 2-8 Anaerobic jar: She is not concerned with the procedure itself, but with the fact that you know how to differentiate between aerobic, anaerobic, facultative organisms based on growth patterns on plates or on growth position in the fluid.

For example, S. aureus is obligate aerobic because there is no growth at all in the anaerobic environment and a heavy growth in the aerobic environment, as well as a thick white layer of growth on the top of the fluid.

1. Aerobic - growth only at top - Bacillus megaterium , alkaligenes faecalis

2. Facultative - growth through out tube but more growth at top - Escherichia coli(anaerobe) , s. aureus(aerobe)

3. Aerotolerant anaerobe - growth throughout tube - Entercoccus faecalis**

4. Anaerobic - growth not at the top of tube where oxygen is present - Clostridium sporogenes

Anaerobic jar:

You will also make streaks of the above cultures on 2 agar plates. One of these plates will be placed in an anaerobic jar and the other will grow under normal oxygen conditions.

Growth on the plates under aerobic or anaerobic conditions will be very definitive for characterizing oxygen requirements of the four cultures.

The facultative organism will grow under both conditions but you should see better growth when oxygen is present.

The aerobic organism will only grow when oxygen is present.

The anaerobic organism will only grow on the plate that was placed in the anaerobic jar.

3-1: Microscopy

Note: ocular lens on top, lightsource=lamp, stage clips to hold the slide in place, condenser under iris diaphragm, stage manipulator=mechanical stage adjustment knob

Base - The part of the microscope that rests on the table is called the base.

Light Source or lamp- The illuminator for your microscope is built into the base and is controlled by an on/off switch.

Arm - The arm is a vertical support that connects the base of the microscope to the body tube.

Ocular lenses - The oculars are the lenses you look through. Some microscopes are monocular--that is, there is only one eyepiece to look through. Other microscopes are dual-viewing; this means there are two oculars--one pointing upwards and one pointing backwards. Yet other microscopes are binocular--that is, there are two eyepieces side-by-side and you look through both of the eyepieces, as you would a pair of binoculars. Most ocular lenses have a 10X magnification.

Revolving Nosepiece and Objective Lenses - Attached to the revolving nosepiece (which is attached to the bottom of the body tube) are several lenses called objectives. Most light microscopes have objective lenses of 4 magnifications:scanning (short) -- normally 4x power, low power (medium) -- normally 10x power, high-power or high and dry (long) -- normally 40x power, and oil immersion (longest) -- normally 100x power.

Stage - The surface on which you place your slide is called the stage. It contains a hole in the center called the stage aperture. Two stage clips or a slide clamp are used to hold the slide in place.

Substage - The area under the stage, called the substage, contains the diaphragm. In addition, some microscopes also have a condenser located under the stage.

o Condenser - The condenser contains a series of lenses that focus light onto the specimen.

o Diaphragm - The diaphragm is an adjustable light barrier built into the condenser that regulates the amount of light passing through the specimen. It is very important that the diaphragm be correctly adjusted in order to get the best possible image. Use the smallest opening that does not interfere with the field of view. The condenser and diaphragm assembly may be adjusted vertically with a knob projecting to one side. Proper adjustment often yields a greatly improved view of the specimen.

Focus Knobs - You can focus your microscope by using the coarse and fine adjustment knobs located on the sides of the arm. Note that the coarse adjustment knob is larger than the fine adjustment knob. The coarse adjustment is used for initial focusing and should be used ONLY when the scanning (4X) objective is in alignment. The fine adjustment makes very slight changes, allowing precision focusing on low, high and dry or oil immersion

Oil between the specimen and the objective lens improves the resolving power. Know why we use oil when viewing microbes at 100x objective: The bacteria are tiny and the oil helps to magnify them.

Know that the total magnification consists of the objective lens(4 scanning, 10 low power, 40 high and dry, or 100 oil immersion) times the ocular lens(10 for our microscope)

We stain in order to provide contrast between the specimen and the background.

3-3 and 3-4: From the 2 page list of organisms: (know the ones above the line as far as where it belongs, ie. Fungus, flagellate, cyanobacteria)—only one question from this—focus more on the ones above the dividing line

Amoeba proteus--protozoa

Anabaena--cyanobacterium

Aspergillus conidiophores—fungi--mold

Coprius mushroom--fungi

Euglena—flagellate protozoan

Fucus male conceptacle—brown algae

Mixed diatoms---single celled algae

Oscillatoria—cyanobacteria—better known as blue-green algae

Paramecium—protozoan--ciliate

Penicillum—fungi/mold

Peridinium--dinoflagellates

Physarium plasmodium—fungus--slimemold

Polysiphonia sporophyte—red algae

Saccharomyces budding cells---fungus/yeast

Saprolegnia—water mold--fungus

Rhizopus sporangia---fungus in soil

Spirogira----green algae

Stemonitis—fungus--slimemold

Volvox—colonial green algae

Saccharomyces—fungus/yeast

Oedogonium macandrous—green algae

Stentor--protozoan

Trypanozoma gambiense—protozoan (looks like a spirochete) cause of african sleeping sickness

Vorticella--protozoan

Ascaris male/female---namatode

Taenia pisiformis gravid--tapeworm

3-5 and 3-6: Know theory section behind simple stains and negative stains

Simple stain: stains are solutions consisting of a solvent(h2o) or ethanol, and a colored molecule(benzene derived), the chromagen. The portion of the chomagen giving off the color is the chromophore. The auxochrome is the charged portion of the chromagen and allows it to

act as a dye through bonds. Basic stains(where the auxochrome becomes + charged by picking up Hydrogen) are attracted to the negative charges of most bacterial cells. Common stains are methylene blue, crystal violet, and safranin. Basic stains are applied to bacterial smears that have been heat fixed, which kills the bacteria and makes them stick to the slide and coagulates the proteins a bit.

Eg…negatively charged cell is being applied a basic stain(it has a positive chromogen). Ionic bond is formed, colorizing the cell.

Steps: add water using sterile loop, add bacteria aseptically, let smear air dry, quickly heat fix, cool off, and add stain, after setting, rinse with water

**Negative stains are used on bacteria too delicate to withstand heat fixing, like the spirochete Treponema, which can be distorted by heat fixing. Negative stain uses a dye solution in which the chromogen carries a negative charge. This would not color the bacteria. It would color the background. The cell remains unstained.

Steps: Add a drop of acidic stain on the corner of a slide, aseptically add organisms on the stain and emulsify with a loop, push the mixture across the slide with a clean slide and dispose of this top slide, air dry and view under microscope (NO HEAT)

3-7: Know how to do a gram stain on p.105(know the stains and removers used—know that it is heat fixed)

Remember the 3 P’s: positive, purple, and peptidoglycan

Before: Prepare smears using aseptic technique. Let air dry and Heat FIX

GRAM STAINING PROCEDURE:

1. Cover with CRYSTAL VIOLET for 1 minute (PRIMARY STAIN)

2. Gently rinse off the stain with distilled water and shake off the excess.

3. Cover with IODINE for one minute.(MORDANT)

4. Pour off the Gram's iodine and rinse with distilled water.

5. Run 95% ETHYL ALCOHOL down the slide until the solvent runs clear (about 10-20 seconds).

(DECOLORIZING AGENT)

6. Rinse with water to stop the action of the alcohol.

7. Cover with SAFRANIN for 1 minute.(COUNTER STAIN)

8. Gently rinse off the stain with water. Blot with bibulous paper

When dry you may view under oil immersion

3-8 Acid Fast stain—also heat fixed. Know the stains and removers used . Know which organisms are stained with this method….Mycobacterium . Why? Because the walls have mycolic acid(waxy surface). Remember, carbolfuchsil is used as the primary stain(red), no mordant is used, and acid-alcohol is used to decolorize here. Brilliant green or methylene is used as the counterstain. Look at p.114

Before: Prepare smears using aseptic technique. Let air dry and Heat FIX

Kinyoun Acid-Fast STAINING PROCEDURE:

1. Cover with Kinyoun Carbolfuchsin for 5 minutes (PRIMARY STAIN)

2. Gently rinse off the stain with distilled water and shake off the excess.

3. Run ACID-ALCOHOL down the slide until the solvent runs clear (about 10-20 seconds).

(DECOLORIZING AGENT)

4. Rinse with water to stop the action of the acid- alcohol.

5. Cover with brilliant green or methylene blue stain for 1 minute.(COUNTER STAIN)

6. Gently rinse off the stain with water. Blot with bibulous paper

When dry you may view under oil immersion

Note: the carbolfuchsin breaks down the waxy mycolic acid layer to allow for staining

3-9Capsule stain procedure—read theory section. This can be used to see certain organisms, such as K. pneumoniae. This is not heat fixed because of the polysaccharides that can be shrinked destroyed by heat. It is a differential stain used to detect cells capable of producing an extracellular capsule. It is similar to a negative stain.

1. Place a single drop of serum and then one drop India ink or Congo Red on the edge of a clean microscope slide.(primary stain)

2. Using a flamed loop and sterile technique, remove some K. pneumoniae (or the organism you want to stain) from your tube or plate and mix it into the drop of stain. 3. Place the end of another clean microscope slide at an angle to the end of the slide containing the organism. Spread out the drop out into a film that goes all the way across the slide. Throw out that top slide.4. Allow the film to air dry. DO NOT heat or blot dry!!!! Heat will melt the capsule!5. Saturate the slide with crystal violet or Maneval’s stain for 1 minute.(counterstain)6. Rinse the slide gently with water.7. Allow the slide to air dry. DO NOT heat or blot dry!!!! Heat will melt the capsule!8. Observe the slide under the microscope, using proper microscope technique.The background will be dark.The bacterial cells will be stained purple.The capsule (if present) will appear clear against the dark background

3-10 Endospore stain: Know that endospores are formed in bacteria for survival under adverse conditions. (not to be confused with fungi spores used in reproduction) You can see them in this stain as dark green dots within red stained cells. This is heat fixed.

1. Perform a bacterial smear of Bacillus or the organism you want to stain, air dry, and heat fix.2. Place a small piece of bibulous paper over the smear. Saturate the paper with malachite green.3. Heat the slide gently over the Bunsen burner for 5-10 minutes. Be sure to keep the bibulous paper saturated with malachite green during heating. If the slide is steaming, you’re okay; if it stops steaming, add more malachite green!4. Remove the bibulous paper from the slide, and rinse the slide gently with distilled water. Dispose of the used bibulous paper in the trash. 5. Counterstain with safranin for 1 minute.6. Rinse the slide gently with water.7. Carefully blot the slide dry with bibulous paper.8. Observe the slide under the microscope, using proper microscope technique.Endospores will stain green. Parent cells will stain red.

3-12: Wet mount p. 122-123

A wet mount preparation is made by placing the specimen in a drop of water on a microscope slide and covering it with a cover glass. Because no stain is used and most cells are transparent, viewing is best done with as little illumination as possible. Motility can often be observed at 10x or 40x, but viewing must be done quick to avoid the moisture from drying up. To look for motility, look for independent darting of the cells. Mass movements while the water is drying up is just a false positive for motility.

1. Place a loopful of distilled water on a slide

2. Add bacteria aseptically to the drop, and flame after transfer.

3. Lower a cover glass over the original slide, making sure there are no bubbles

4. Observe at 40X or under 100X, and dispose of slide when done

4-5 and 4-6: MacConkey: to detect members of the Enterobacteria, which happen to be gram negative lactose fermenters, like E. Coli and Klebsiella Pneumoniae and Enterobacter

EMB: differential medium used to isolate fecal coliforms, to detect members of the Enterobacteria, which happen to be gram negative lactose fermenters, like E. Coli and Klebsiella Pneumoniae and Enterobacter.

On MacConkey, the positive lactose fermenters turn fuschia. Negative stays the color of the medium(clear like)

On EMB, the positive lactose fermenters(Klebsiella and Enterobacter) turn pink to purple, and in addition, E.Coli turns metallic green. Negative(which is non lactose fermenting or sucrose fermenting) remains the color of the medium(clear like)

5-28 Motility: Through the stab method in agar in a tube, motile appears to have areas diffusing away from stab line, while non-motile retains the solid line. One a plate, one would see waves or concentric circles for motile

EG. Klebsiella—nonmotile, while E.Coli and Proteus motile

12-1: Identifying fungi—

p.398 molds are fuzzy on plate

P.398: visually identify yeast in microscope by its budding nature of reproduction(binary fission) Also, they are bacteria like in shape, but dull and dry looking on plate. They do not glisten, since they are kind of waxy

Penicillum is fungi but a mold type p.404 both pictures . The colony on agar looks green and granular, and under a microscope, its condiophores look like brooms

Know what Aspergillus conidia looks like p.403—looks like it has peacock feathers

p. 402—top left picture for rhizopus—looks like flowers with a cottony topping