Download Lab Practical 1 Detailed Review

Lab Practical 1 Review
1. What treatment is performed on workstations before and after lab sessions and why?
A.) The work station is wiped down with wescodyne to disinfect the area.
2. Why are workstations kept free of clutter?
A.) To keep them from becoming contaminated or contaminating the disinfected work station.
3. Proper Bunsen burner usage:
A.) Shut off when unattended
Tie back long hair
Don’t share
4. Why are food and drink prohibited in the lab?
A.) To prevent them from becoming contaminated and then consumed.
5. Why do you need to wear a lab coat?
A.) To prevent contamination and stains to clothes and to prevent any microbes on clothes from
contaminating specimens. It is left in the lab room to prevent any microbes from experiments
from leaving the lab room and contaminating things outside.
6. Why are gloves worn in the lab?
A.) They are worn to prevent contamination to ourselves from specimens and to prevent any
microbes on our hands from contaminating specimens. Always wash hands at the end of lab to
remove any transient microbes or others you may have picked up during your lab experiment.
7.
a). Used test tubes get returned to their designated rack or bin.
b). Broken glass needs to be disposed of in a Sharps container.
c). Plates get disposed of in the designated biohazard bin.
8. Why must cultures never leave the lab?
A). So as to prevent spreading the microbes used in experiments which could cause illness.
9. Know the location of the fire extinguisher, eye wash station, fire blanker, emergency kit, etc.
10. Properly disinfect bacterial spills by cleaning with wescodyne. Also, notify your professor.
11. Best way to prevent accidents?
A). Read and understand the lab procedure before coming to class.
Exercise 1 (Use of Microscope)
1.
a). resolution: The ability of a lens system to distinguish as separate entities objects that are
very small and very close together. (Determined by quality of lens and the wavelength of light
used.)
b). magnification: making a small object appear larger.
c). parfocal: maintaining focus while switching magnifications on the microscope.
d). parcentric: a microscope that has the ability to rotate between objectives without the subject
shifting from the center of the field of view. Acceptable parcentricity is to within 1/3 of the field
of view.
e). depth of field: the ability to focus at different planes in the specimen b/c of thickness (of
specimen). Decreases as magnification increases.
f). field of view: the portion of the slide that is visible in the circle of light.
g). working distance: distance between the objective lens and the stage.
2. Know the functions and parts of a microscope and be able to label them:
3. Know proper care and cleaning of a microscope.
4. Know proper focusing procedures from start to finish:
A). Always start at 4x then work your way up, refocusing as needed with the fine adjustment
knobs.
5. Be able to calculate total magnification:
A). Objective: 4x (scanning lens) - Ocular magnification: 10x – Total magnification = 40x
Objective: 10x – Ocular magnification: 10x – Total magnification = 100x
Objective: 40x (high dry) – Ocular magnification: 10x – Total magnification = 400x
Objective: 100x (oil immersion) – Ocular magnification: 10x – Total magnification = 1000x
6. Purpose and procedure of oil immersion?
A). It helps minimize light scatter to see an image more clearly.
7. Know proper storage procedure for the microscope:
A). Middle row of microscope goes stage out, all the way up, magnification on 4x. Clean,
remove all slides, and wrap up cord.
Exercise 2: (Aseptic Technique)
1.
a). aseptic: without contamination.
b). inoculation: transferring a microbe from one medium to another.
c). inoculum: the sample that is being transferred during inoculation.
d). turbidity: cloudy
2.
a). inoculating loop: the tool used to transfer samples during inoculation.
b). test tube: houses liquid and other types of medium for inoculating.
c). petri plate: used to grow colonies of microorganisms.
3. Know how to sterilize a loop using a Bunsen burner.
4. Know how to aseptically transfer cultures from a test tube or plate, to a slant, broth, and plate.
5. Why is aseptic technique so important?
A). It prevents contamination of cultures (from you and environment). Also of you and the
environment (from cultures).
6. Know how to properly label test tubes and plates.
7. Know proper procedure for disposal of used test tubes and plates.
8. Understand how to use colony morphology (growth patterns) to distinguish organisms as being
separate species.
9. Know how to observe growth patterns in broth cultures:
a). turbidity: cloudiness
b). flocculent: chunks floating around
c). sediment: on bottom of tube
d). pellicle: film of bacteria covering the surface of the liquid
e). ring: growth only around the edge (no surface growth)
Exercise 8 (Simple Staining and Smear Prep)
1.
a.) simple stain: uses only one kind of stain. Can be either positive (bacteria is stained) or
negative (background is stained, bacteria appears bright white). Safranin, methylene blue, or crystal
violet. The purpose of a simple stain is to observe the morphology and arrangement of bacterial
cells.
b). morphology: the shape and arrangement of cells i.e., coccus (round), bacillus (rod), spirillum
(spiral), vibrio (comma).
2. A chromogen (chromophore) is used in simple staining. Why?
A). A stain with a positive chromogen will stain the bacteria in a simple stain, not the
background. This is b/c bacterial nucleic acids and certain cell wall components carry a negative charge
that strongly attracts/binds to the chromogen. THIS WILL ALLOW FOR THE MORPHOLOGY
AND ARRANGEMENT OF THE CELLS TO BE OBSERVED.
3. Know the purpose and procedure of a simple stain and what stains are used.
A). To observe the morphology and arrangement of bacterial cells. Safranin, methylene blue, and
crystal violet can be used in a simple stain.
4. Know and identify the 4 most common shapes of bacterial cells:
A). Coccus: round
Bacillus: rod
Spirillum: spiral
Vibrios: coma
5. Know the different cell arrangement prefixes and identify them:
A). single, diplo, tetrad, strepto (chain), staphylo (cluster).
6. Know why too thick of a smear is a problem when staining and observing cell shape and arrangement:
A). There will be huge clumps of bacteria and the staining may be uneven. Cell shape and
arrangement will be difficult to view. Also, the bacteria may not heat fix.
7. Know how to properly create a smear from a broth culture or slant culture:
A). Dip loop in (after sterilizing), obtain sample, make a zig-zag pattern on the agar in the dish,
lightly.
8. Know how to heat fix a slide and its purpose:
A). Run the slide quickly over the flame on the Bunsen burner 2 to 3 times. Do this to kill the
bacteria and adhere it to the slide so it does not get washed off during staining.
9. Know how to stain and rinse a slide.
Exercise 9 (Negative Staining)
1. Chromophore (chromogen): an agent in the dye that is either positively or negatively charged,
depending on the kind of stain.
2. An acidic chromogen is used in a negative stain b/c:
A). Bacteria are acidic and have a negative charge. The dye will stain the background on the
slide and not the bacterium because it will be repelled by the negative charge of the cell (-,- repel). This
is used for bacteria with sensitive structures like flagellum.
3. Procedure and purpose of a negative stain:
a). Add a drop of nigrosen to one end of the slide, not in the center.
b). Aseptically add a small amount of one bacterium to the drop.
c). Flame loop
d). Aseptically add a small amount of another kind of bacterium to create a mixed slide.
e). Pick up a second clean slide, use this slide to spread the smear.
f). Start on the end of the slide, away from the dye. Slide the edge and across the width of the
slide.
g). Quickly pull the second slide down the length of the slide. This should result in a long, dark
smear with varying thickness along the entire length of the slide.
h). When dry, cover with a cover slip and view under oil immersion.
Purpose: cells will be bright white against the dark bakground and b/c negative stains are not
heat fixed (due to the sensitive nature of the bacteria), cells retain their morphology characteristics.
(Preserves Structure)
Exercise 10 (Capsule Stain <Differential Stain>)
1. Glycocalyx: (“sugar coating”) General term for an extra cellular (outside cell) structure produced by
some bacteria. The slimy layer on a fish can be considered a glycocalyx.
- If it is firmly attached to the cell wall, it is called a capsule.
- If it is less compact and loosely attached to the cell wall, it is called a slime layer.
a). Capsule: Composed of polysaccharides and/or polypeptides (amino acids).
- energy and water reserves (storage), helps the cell remain hydrated.
- helps evade phagocytosis and the immune system.
- serves as an anchor or surface attachment mechanism because it is “sticky”.
- has definite boundaries, not easily washed off.
- in less than favorable conditions, bacteria can metabolize the molecules in their capsule
for energy.
b). Slime layer: can be easily washed off.
2.
a). Purpose of a capsule stain: (It is a differential stain) To reveal the presence of the bacterial
capsule. The capsule is often difficult to see with simple staining or after a gram stain.
b). Procedure:
- aseptically add one loop full of culture to a slide
- spread the inoculum so that your smear is about the size of a nickel. This smear should
also be thicker than other smears you’ve done.
- Air-dry. DO NOT heat fix, it will melt the capsule.
- cover with the primary stain (can be either crystal violet or safranin) for 1 minute.
- rinse the slide with copper sulfate for 20-30 seconds (do not use water)
- blot dry and observe under oil immersion
3. Be prepared to identify a capsule stain and label the bacterium and capsule from a picture.
Exercise 11 (Gram Stain <Differenetial Stain>)
Gram + cells have a thick peptidoglycan cell wall
Gram – cells have a thin peptidoglycan cell wall and a lipopolysaccharide (LPS, endotoxin) outer membrane.
1.
a). primary stain: (crystal violet) applied to the heat-fixed slide and sits for one minute. This is a
basic stain that dyes all cells on the slide purple.
b). mordant: (iodine) pour Gram’s iodine and let sit for 1 minute. This (mordant) fixes color into
the cell or intensifies it. It binds to the RNA and crystal violet in the cell and creates a larger color
complex.
c). decolorizer: (alcohol) rinse slide for 20-30 seconds. Alcohol is a solvent/denaturant. It
creates large holes in the gram-negative cell’s outer lipopolysaccharide cell membrane and washes out
the iodine-crystal violet complex. The thick peptidoglycan layer in gram-positive cells retains the
iodine-crystal violet complex. **After this step, gram-neg cells are clear and gram-pos cells are
purple.**
d). counterstain: (safranin) applied for 1 minute. Then rinse with water. Gram + will be purple
and Gram – will be pink or red.
2. The purpose of a differential stain is:
A). it differentiates, by color, two major groups of bacteria. Includes a primary stain and a
counterstain.
3. Know how cell walls differ in Gram + and Gram – cells:
- Gram-positive: have a thick layer of peptidoglycan and DO NOT have an outer cell membrane.
- Gram-negative: have a thin peptidoglycan cell wall but also have a lipopolysaccharide outer
cell membrane.
4. Know the color of the cells after every step in a gram stain:
a). crystal violet – both cells are purple.
b). gram’s iodine – both cells are still purple.
c). alcohol – gram-neg is colorless and gram-pos are still purple.
d). safranin – (counterstain) gram-pos are still purple and gram-neg are pink or red.
Exercise 12 (Acid-Fast Stain <Differential Stain>)
1. Purpose of the acid-fast stain:
A). Differentiates between acid-fast and non-acid-fast bacteria.
- Ziehl-Neelsen carbol fuchsin (primary stain) is used, acid alcohol is then used to
decolorize, and counterstain with methylene blue. Steam is also used and is important for creating
fluidity of the waxy cell wall.
2. Acid-fast bacteria include the genera Mycobacterium and Nocardia.
3. Acid-fast cells have a waxy cell wall. Mycolic acid in the cell walls of acid-fast bacteria makes these
organisms resistant to desiccation and disinfectants. It also makes them difficult to stain.
4. Be prepared to identify an organism as acid fast positive or negative in an image. Cells that retain the
primary stain after decolorization are called acid-fast and are bright red/fuschia. The secondary stain
(methylene blue), is then applied to stain the non-acid-fast cells. These cells will appear blue.
Exercise 13 (Endospore Stain <Differential Stain>)
1.
a). vegetative cells: active/growing/metabolizing cells.
b). sporulation: the beginnings of forming a spore and starts with the replication of the bacterial
chromosome and the spatial separation of the chromosomes in different regions within the cell. Occurs
when vegetative cells are stressed by such factors as nutrient limitation, dehydration, temperature
change, and waste accumulation.
c). endospores: survival technique of certain bacteria that help them survive harsh, unstable
environmental conditions until more favorable, stable conditions return.
d). anaerobe: an organism that does not require oxygen for growth.
e). aerobe: an organism that requires oxygen to grow.
2.
a). Purpose of an endospore stain:
- To differentiate between species of bacteria that can produce endospores and those that cannot.
b). Procedure:
- Prepare a slide of bacteria and heat fix it.
- Place slide on staining tray.
- Put a piece of pre-cut blotting paper on top of the slide.
- Flood the entire slide with Malachite green and leave on for 50 minutes. Make sure it
does not dry out so check periodically and reapply more stain if needed.
- Remove blotting paper and rinse well until water runs clear.
- Flood with safranin for 1 minute.
- Allow the slide to dry and do not blot with paper towel.
3. Be prepared to identify an endospore stain with vegetative cells and endospores in a picture:
4. The two bacteria that produce endospores by genus: Remember to capitalize and italicize (or
underline) the Genus.
a). Bacillus
b). Clostridium
5. Know how a 24 hour culture will differ from a week old culture and why:
A). the week old culture will have more endospores as the environment becomes more and more
unfavorable and the vegetative cells need to commence sporulation.
Exercise 29 (Parastitc Protozoa)
1. Know the 2 cellular states of protozoans, how they differ and what causes or triggers a particular
state:
a). trophozoite: this is the active, motile, reproductive state. Protozoans are in this state most of
the time and when environmental conditions are favorable.
b). cyst: this is a dormant state that protozoans enter when conditions become unfavorable like
lack of food and moisture. This state is tough and resistant. This state is often involved in disease
transmission.
2. Be prepared to name and identify the following:
a). Giardia lamblia (cyst and trophozoite):
Cyst
Trophozoite
b). Trypanosoma gambiense (trophozoite): (will be seen with red blood cells)
c). Trichomonas vaginalis (trophozoite):
d). Entameoba histolytica (cyst and trophozoite):
e). Balantidium coli (cyst):
f). Toxoplasma gondii (trophozoite):
g). Plasmodium falciparum (ring and schizont stage) (will be seen in red blood cells):
Exercise 30 (Parasitic Worms)
1.
a). dieocious: A species that has both male and female individuals (like humans).
b). monoecious: a species that on one organism can exist male and female parts
(hermaphroditic).
c). intermediate host: the host in which larval development occurs.
d). definitive host: the host in which adulthood and mating occur.
e). parasite: an organism that lives on or in a host organism and harms that organism.
2. Be prepared to identify the phylum for each of the worms observed:
a). Phylum Platyhelminths – flat worms, i.e., flukes and tapeworms
b). Phylum Nematoda – round worms i.e., pin worms, Ascaris, hookworms.
3. Know the method of reproduction for all worms observed:
a). Roundworms are dioecious and reproduce sexually. They have a complete digestive system
with two openings. They use eggs for reproduction.
b). Flatworms are monoecious and are hermaphroditic. They have a primitive digestive system
with only one opening for eating and waste elimination.
4. Be prepared to identify the Chinese Liver Fluke, (Clonorchis sinensis), adult and egg. Also identify its
testes, ovaries, and eggs.
5. Be prepared to identify the eggs of the lung fluke (Schistosoma mansoni):
6. Be prepared to identify an adult tapeworm (Taenia is the Genus name) and the eggs of any Taenia
Genera:
b). function of scolex: hooks/suckers that help it hold onto the host’s tissue.
c). function of the proglottids: segments on a tapeworm that have both male and female
reproductive organs.
7. Be prepared to identify the hookworm (Necator americanus) eggs:
8. Be prepared to identify the pinworm (Enterobius vermicularis) adults and eggs:
9. Be prepared to identify the giant roundworm (Ascaris lumbricoides) adults and eggs:
10. Be prepared to identify Trichinella spiralis (larvae) encysted in muscle tissue: