What does TSA stand for?
tryptic soy agar
What four areas did we swab in Exercise #1?
The surface of the tongue, crevice b/w nose and face, unflushed commode, and aluminum hand rail.
What temperature is the incubator set at?
What is the definition of the aseptic technique?
lab procedures to prevent contamination
What are some examples of aseptic techniques?
Flaming your loop, using the disinfectant for your lab area, washing your hands before you leave lab, and the foot stirups used at the sink.
What are the two important things about safety?
1. Don't reach across flame to get something. Make sure the burner is pushed far away from you.
2. Wear gloves if you have cuts or scrapes. Also, don't put your hand to your mouth.
What are the only glass products that go into the big glass jar on lab tables?
Pasteur pipettes and slides.
How many quadrants do you use for a streak plate?
T or F. There are very few places that bacteria can not be found.
What factors influence the types and numbers of bacteria found in different places?
cleaning frequency, temperature, chemical toxity (metal, disinfectants, antibiotics), presence or absence of moisture, exposure to radiation(UV in sunlight), exposure to wind currents, presence or absence of growth supporting nutrients, accessibility to contamination from the enviroment.
Why is tryptic soy agar useful?
It is relatively inexpensive and many microbes are cabable of growing on it.
What is tryptic soy agar composed of?
Digested soybeat protein, some essential inorganic salts, water, and a gel-like compound called agar.
What is capable of growing on TSA?
molds, bacteria, and yeast
What is not capable of growing on TSA?
Protozoa, algae, viruses and certain bacteria, molds, and yeast.
Microbes that grow on the TSA are called what?
What two colonies are often difficult to distinguish on TSA?
Yeasts and bacterial colonies.
What is usually distinctive to distinguish on TSA?
Molds (large, cottony, multi-colored, spreading, above agar colonies)
What is 3 goals of making a streak plate?
1. To obtain isolated bacteria so that a pure culture can be obtained and verified. 2. Enable one to obtain additional (and younger) bacteria for further invesigation. 3. Help one estimate the approximate diversity and numbers of microbes found at a particular site.
When looking at a streak plate, what are some things you can identify them by?
Colny's margin, configuration, and elevation. Pigmentation and colonial consistency (mucoid, butyrous, dry and/or adherent).
What color is gram negative? Positive?
What are the tubes of agar called where the gel is angled?
How often should you make a new culture?
Once every two weeks
What is the term called that are made as intial steps to staining bacteria?
How should the smear look?
Slightly turbid. If it appears "milky" there are too many bacteria!!
What are the two most important steps in smears?
air-dried and heat-fixed. Not doing this will cause the organisms to be washed from the slide during staining. Avoid excessive heating though.
On the basis of the Gram stain, bacteria can be classified into what two groups?
gram positive or gram negative.
What is the primary stain and what color does it stain all bacteria?
Crystal violet. It stains all bacteria blue.
What is the mordant (fixant) and what does it do?
Iodine. It reacts with the crystal violet to fix the blue color w/in the cell.
What is the decolorizer and what does it do?
95% ethanol. It will extract the stain from the gram negative cells but not gram positives.
What is the counter-stain and what does it do?
Safranin. It stains all decolorized cells red while leaving nondecolorized cells blue.
What is the difference in gram + and gram -?
The kind of cell wall that a bacterium produces. Because the cell wall degenerates w/ age, one should not rely on the results of a gram stain done with on a culture which is over 24 hours old. Gram + cell walls are especially subject to degeneration over time and the cells can appear gram -.
What is another gram stain called that shows gram - and gram + results?
What can cause gram variable?
Presence of old/dead cells, a culture that has been exposed to wall damaging materials such as anti-microbial drugs, a culture that is easily decolorized, or a stain that has either has been over or under decolorized. Also, it can result from an uneven stain penetration when a smear is prepared with too many cells.
T or F. Bacterial spores and capsules seldom retain stain and will appear colorless with the gram stain method.
T or F. Although one can determine cell shape by looking at the gram stain results, it is more reliable to use a wet mount for this purpose.
T. or F. The heat-fixing step used in the gram stain procedure will sometimes shrink short rods to the point which they are mistaken for spheres (cocci)
What is a capsule?
A carbohydrate coating with a very high water content that some bacteria produce. It may be regular, irregular, loosely attached or tightly attached but ALWAYS extracellular.
What is the purpose of a capsule?
It may be for protection, for adherence or as a hedge against dehydration. Capsules usually will not stain and therefore negative stains are necessary to view them.
What is the ink called that is composed of numberous dark carbon particles that is used to stain the background?
India Ink. It will not penetrate the capsule and therefore the capsule is left unstained and will appear colorless. Hence, a positive test is evidenced by a blue cell, surrounded by a clear zone (capsule) w/in a dark brown background.
Why are TSA slants typically used to store microbial cultures?
They take up less storage space, the medium won't dry as quickly when kept in a properly capped test tube compared to medium in a pertri dish, and one is also less likely to experience contamination from the air when working from a slant b/c the entrance to the slant is smaller.
Why is the TSA slant slanted?
To increase surface area so that more bacteria can be produced.
What are the 3 extracellular enzymes we tested in Unknown lab #3?
Amylase. If + then it carries out starch hydrolysis
Lipase. If + then it carries out lipid/fat
Gelatinase. If + then it carries out gelati
hydrolysis. May also be termed
proteolytic (protein hydrolysis)
What agars were used with amylase, lipase, and gelatinase?
amylase=starch agar (large carbohydrate), lipase=tributyrin agar (lipid), and gelatinase= gelatin agar (protein).
What was the only enzyme test that no reagent was required on the extracellular enzymes?
What did you flood the plate with during the amylase test? Gelantinase test?
We flooded the plate with iodine; waited 5 minutes, then examined for a clear zone around the growth. We flooded the plate with saturated ammonium sulfate for the gelantinase test. We waited 10 min. then examined plate for clear zone around growth.
What are the 4 intracellular enzymes?
Tryptophanase, urease, catalase, and oxidase.
What were the only 2 intracellular enzyme tests that we knew right away?
Catalase and oxidase.
Give a description of the tryptophanase test.
We used a SIM medium (sulfur indole motility) tube. We stabbed it w/ a needle and streaked the surface. The next lab we looked for diffusion of growth from the stab line which indicates possible motility. A distinctive black pigment in the medium should be recorded as hydrogen sulfide (H2S). Then we added 1/4 inch Kovac's reagent and waited at least 1 min. but no longer than 5. A red color indicates it is indole positive.
Give description of the urease test.
We used urea broth and scraped a visible amount of our unknown into the urea broth. The next lab we looked for a red-orange-rose color change for a positive test.
Describe the catalase test.
We used the streak plate from last week from the frige. We placed one drop of 30% hydrogen peroxide directly on bacteria. Formation of bubbles w/in 1 min. is positive for catalase.
Describe the oxidase test.
We scraped a visible amount of bacteria into a Dry Slide oxidase "window". A purple color indicated it was positive for oxidase. This test should be performed with YOUNG bacteria.
Describe the spore stain.
We prepared a smear of our unknown from the slant tube. We then used the Schaeffer-Fulton method. (Know this method; don't have the manual so can't write it yet) Your cells should be stained red.
If your cells are producing spores, what color should they be?
How do you do a wet mount?
You put a drop of water on a slide then scrape bacteria fromt the slant tube. Mix the bacteria in the water until you have a SLIGHTLY CLOUDY mixture.
What should you use is you place a coverslip on your wet mount and you have excess water coming out of the edges?
Use bibulous paper
T or F. When doing a wet mount, you need to examine the preparation IMMEDIATELY.
How far away is the distance b/w any 2 adjacent marks on the micrometer scale in a microscope?
T or F. You record length and diameter for an average, small, or large sized cell and ONLY the diameter for cocci.
What can inclusion bodies include?
Spores, energy or carbon storage compounds.
What is the definition of extracellular enzymes?
Enzymes produced w/in the bacterial cell which are then released to the outside. They break large substances into smaller substances which allows the cell to bring them inside to be used as a food source.
what is the definition of intracellular enzymes?
Enzymes whose function is carried out inside the cell.
What is starch agar composed of?
Beef extract, peptones, agar, and soluble starch.
Amylase breaks starch down into what components?
Starch---> Dextrins, Maltose, and Glucose
What color results from the interaction of starch and iodine?
black. Ind-products of its degradation (maltose and glucose) don't react w/ iodine. The result is that in those areas of the meduim where starch has not been broken down the meduim will stain dark and in those areas where the starch has been degraded the medium won't stain and therefore remain transparent and colorless after flooding the plate w/ iodine.