If your instructor uses powerpoints use those and write your notes on them. Flashcards are very helpful. I used colorful notecards and printed colored pictures and pasted them onto my notecards. After doing so I would write the info on the other side. I showed my instructor my notecards and he was very impressed and thought I should sell them. I was shocked. I keep everything.
You should also find yourself a study group. I found that very helpful while taking that class. I thought yeah, I can do this alone, like I always do. I got a 68% on my first test. I broke down and cried. After using the powerpoints, flashcards, and study groups...I made it out of Micro with a B. If only I would have gotten a higher grade on my first test I would have made it out with an A. I kept kicking myself in the butt. I got A's on papers, homework, and two other exams. Ugh.
I'm also going to post some notes I have on my laptop that my group made that we felt was helpful....and maybe it will help you.
Biosafety in Microbiological and Biomedical Laboratories
Organisms do not typically cause disease
Handled in open
No special containment equipment is required
Ex.: Bacillus subtilis, Escherchia coli, Rhodospirillum rubrum, and Lactobcillus acidophilus
Organisms are commonly encountered in the community
Cause human diseases
Ex.: Salmonella, Staphylococcus aureus, Clostridium dificile, and Borrelia burgdorferi
Local or exotic origin
Associated with respiratory transmission and serious or lethal disease
Personnel handle microbes in a Class I or II biological safety cabinet (BSC), not on the open bench.
Ex.: Bacillus anthracis, Mycobacterium tuberculolosis, and West Nile virus.
Organisms have a great potential for lethal infection
Ventilation and waste management
Specially trained personnel perform transfers in Class III BSCs.
Wear positive pressure, one-piece body suits with a life-support system.
Ex.: hemorrhagic diseases, such as Ebola virus, Marburg virus, and Lassa fever
Do not smoke, eat, or drink, or bring food or drinks into the lab
Do not apply cosmetics or handle contact lenses
Wash your hands before and after
Do not take organisms out of lab
Come to lab prepared
Do not rush in lab
[*]Basic Laboratory Safety
Wear protective clothing
Do not wear sandals or open-toed shoes
Wear eye protection when heating chemicals
Turn off your Bunsen burner when t is not in use
Tie back long hair
Feeling ill, go home
If you are pregnant, immune compromised, or are taking immunosuppressant drugs, please see the instructor
Take off and dispose gloves. Wash hands
Wear gloves when staining and handling blood
Use antiseptic if you are exposed to a spill
Never pipette by mouth
Dispose of broken glass or any other item into the appropriate sharps or broken glass container
Use a fume hood when using chemicals or stains that need to be heated
Find the first-aid kit, and make a mental note of its location
Find the fire blanket, shower, fire extinguisher, and eye wash basin. Know where the locations are and how to use them.
[*]Reducing Contamination of Self, Others, Cultures, and the Environment
Wipe the desktop with a disinfectant before and after
Never lay down culture tubes on the table; upright in a tube holder
Cover any culture spills with paper towels. Soak the towels immediately with disinfectant, and allow them to stand for 20 minutes. Report the spill to your instructor. Place the towels in the container designated for autoclaving.
Place all nonessential books and papers under the desk.
When pipetting, place a disinfectant-soaked towel on the work area. This reduces contamination and possible aerosols if a drop escapes from the pipette and hits the tabletop.
[*]Disposing of Contaminated Materials
Remove all labels and place them in the autoclave container so designated
Dispose of plate cultures. Petri dishes should be taped down. Put in correct disposal.
Dispose blood and gloves in correct bin.
Microscope slides in the correct bin.
Contaminated broken glass and other sharp objects in a sharps container for autoclaving.
Uncontaminated broken glass can go to a broken glass container.
Look at page 70 in lab book for overview of microscope (Know all the parts of the microscope)
Refraction: (bending) of light as it passes through the objective lens from the specimen produces a magnified real image.
Resolution: clarity of an image. Is the ability to disguised objects that are close together.
Parfocal: A parfocal lens is a lens that stays in focus when magnification/focal length is changed. There is inevitably some amount of focus error, but small enough to be considered insignificant.
3-3 Wet Mounts
Look at 3-13, 3-16, 3-17, 3-18, 3-19 (Don’t memorized species, just yeast, green algae, etc)
Figures 3-13: Amoeba, A Sarcodine
Figure 3-16: Conjection of pili’s
Figure 3-17: Vovox; Green Alga
Figure 3-18: Yeast
Figure 3-19: How to make a wet mount
Prokaryotes: The prokaryotes are a group of organisms that lack a cell nucleus, or any other membrane-bound organelles. Domains Bacteria and Archae. 1-5 um. Ribosomes 70S.
Eukarytoes: nucleus. Has both nonmenbrane bound and membrane bound organelles. Domain Eukarya. 10-100um. Ribosomes 80s.
Tropozoite: the form of a sporozoan protozoan in the feeding stage. (Vegetative state)
Cyst: A microbial cyst is a resting or dormant stage of a microorganism, usually a bacterium or a protist or rarely an invertebrate animal, that helps the organism to survive in unfavorable environmental conditions.
Pseudopods: A temporary projection of the cytoplasm of certain cells, such as phagocytes, or of certain unicellular organisms, especially amoebas, that serves in locomotion and phagocytosis.
Green Algae: Any of various photosynthetic protists belonging to the phylum Chlorophyta. The green algae share many characteristics with plants, notably in their having chlorophylls a and b, in their storage of food as starch, and in the composition of their cell walls from cellulose or other polysaccharides. The green algae show a great variety of body types, ranging from unicellular forms to filaments to leaflike thalli, and many species live in colonies. Green algae also show a variety of reproductive processes, both sexual, by the formation of conjugating gametes or the exchange of nuclei through conjugation tubes, and asexual, by means of spores. Green algae are mostly aquatic, in both freshwater and marine environments. However, many species live on land or in the soil, and even in extreme environments, such as the surface of snow. Green algae are not always green, since they produce carotenoid pigments that can give them orange or red colors. Some lichens consist of a symbiotic relationship between a fungus and a green alga.
Absorptive: (Fungi) fungi feed by absorption of nutrients from the environment around them. They secrete exoenzymes into their environment, then absorb the digested nutrients. Most are saprophytes that decompose dead organic matter, but some are parasites of plants, animals, or humans.
Heterotrophs: an organism deriving its nutritional requirements from complex organic substances.
Exoenzymes: an enzyme that acts outside the cell that produces it.
Saprophytes: a plant, fungus, or microorganism that lives on dead or decaying organic matter.
Parasites: an organism that lives in or on another organism (its host) and benefits by deriving nutrients at the host’s expense.
Yeasts: a microscopic fungus consisting of single oval cells that reproduce by budding, and are capable of converting sugar into alcohol and carbon dioxide. (Fermentation)
Molds: a typically multicellular fungus that grows as long filaments called hyphae and reproduces by means of spores.
Wet mount: a small flat rectangular piece of glass on which specimens can be mounted for microscopic study. (Figure 3-19)
1-3 Page 17 AsepticTransfers and Inoculation Methods
- Figure1-8 Know all the instruments
- Figure 1-10 Bunsen Burner Flame (Outer cone andInner cone)
1-4 Streak PlateMethods of Isolation
Mixed Culture: a microbial culture consisting of two or more species.
Pure culture: contains only a single species.
Isolation technique: four quadrants of the streak plate. The streaking is done using a sterile tool, such as a cotton swab or commonly an inoculation loop. This is dipped in an inoculum such as a broth or patient specimen containing many species of bacteria. The sample is spread across one quadrant of a petri dish containing a growth medium, usually an agar plate which has been sterilized in an autoclave. To get colonies.
Streak plate: a streak plate method of isolation, a bacterial sample is streaked over the surface of a plated agar medium. Used to get isolation.
Colonies: consisting only of the original cell type.
Colony-forming unit (CFU): a single cell or group of related cells that produce a colony.
[h=1]2-1 Ubiquity of Microorganisms[/h]
Free-living: microorganisms do not reside on or in a specific plant or animal host and are not known to cause disease. In other words, they live freely and independently, not as a parasite or attached to a substrate.
Host: an animal or plant on or in which a parasite or commensal organism lives.
Nonpathogenic: Incapable of causing disease. For example, nonpathogenic E. coli are E. coli bacteria that do not cause disease, but instead live naturally in the large intestine.
Pathogens: is a microorganism such as a virus, bacterium, prion, or fungus, that causes disease in its animal or plant host.
Commensal (Commensalism): a form of symbiosis in which one organism derives a benefit while the other is unaffected, does not benefit, or get harmed.
Symbiosis: Prolonged association between two or more organisms of different species.
[*]Mutualistic (Mutualism): symbiotic interaction between different species that is mutually beneficial.
[*]Opportunistic: denoting a microorganism that does not ordinarily cause disease but that, under certain circumstances becomes pathogenic.
[*]Opportunistic pathogens: those that take advantage of certain situations—such as bacterial, viral, fungal or protozoan infections that usually do not cause disease in a healthy host, one with a healthy immune system. A compromised immune system, however, presents an "opportunity" for the pathogen to infect.
[*]Even many of the commensal or mutualistic strains inhabiting our bodies are opportunistic pathogens.
2-2 Colony Morphology
Figure 2-3: Colony morphology (I noticed that some of the terminology is not shown in this figure).
Figures 2-4 through 2-24: Use to study the colony morphology terminology.
Punctiform (tiny, pinpoint): having the form of a point.
[*]Margin: The margin or edge of a colony.
Undulate : wavy
Filamenous: in the form of very long rods, many times longer than wide.
Rhizoid: branched like roots
[*]Elevations: The description of the “side view” of a colony.
Mucoid: relating to or resembling mucus; “a mucoid substance”
Opaque: Not transparent or translucent; impenetrable to light; not allowing light to pass through.
Translucent: permitting light to pass through in a diffuse manner.
Page 95 BacterialStructure and Simple Stains
Figure 3-72 Division patterns among cocci
Figures 3-64 through 3-78 use to practice applying cellular arrangement and morphology terminology.
Contrast: The difference in visual intensity between two objects or between an object and its background.
Arrangements: What and where things are on the bacteria.
Cocci (singular coccus): can be used to describe any bacterium that has a spherical shape.
Bacilli (singular bacillus): rod-shaped bacterium.
Spirilla (singular spirillum): spiral-shaped bacterium.
Vibrios: slightly curved rods
Coccobacilli: short rods
Spirochetes: flexible spirals
Pleomorphism: a concept whereby cells take on multiple structural forms.
Diploccus: if the two daughter cells remain attached after a coccus divides.
Diplobaccilli: if the two daughter cells remain attachter after a bacilli divides.
Streptococuss: if the coccus cells keep dividing in the same plane and remain attached.
Streptobacillus: if the bacilli cells keep dividing in the same plane and remain attached.
Tetrad: if a second division occurs in a plane perpendicular to the first. Only seen in cocci
Sarcina: a third division plane perpendicular to the other two produces a cube-shaped arrangement of eight cells. Only seen in cocci
Staphylococcus: if the division planes of a coccus are irregular. A cluster of cells.
3-5 Simple Stains
Figure 3-79 Chemistry of Basic Stains
Figure 3-80 Safranin Dye in a Simple Stain
Figure 3-81Heat fix
Chromagen: any substance found in organic fluids that forms colored compounds when oxidized. It is the part of the stain that colors the cells.
Basic stain: are attracted to the negative charges on the surface of most bacterial cells. Positive stain. Thus, the cell becomes colored. Ex.: methylene blue. safranin, crystal violet
Heat-fixed: basic stains are applied to bacterial smears that have been heat-fixed. Heat-fixing kills the bacteria, makes them adhere to the side, and coagulates cytoplasmic proteins to make them more visible. It also distorts the cells to some extent.
3-6 Negative Stains
Figure 3-83 Chemistry of acidic stains
Figure 3-84 A Nigrosin Negative Stain
Figure 3-85 Procedure for negative stain
Acidic stain: the negative staining technique uses a dye solution in which the chromogen is acidic and carries a negative charge. The negative charge on the bacterial surface repels the negatively charged chromogen, so the cell remains unstained against a colored background.
3-7 Gram Stain (differentialstain)
Figure 3-86 Gram Stain steps
Figure 3-87 Gram stain example (both positive and negative gram stain)
Figure 3-95 Procedural diagram: gram stain
Decolorization: remove the color from (alcohol)
Primary Stain: the first stain (crystal violet)
Mordant: a substance that binds to a dye and makes it less soluble (Iodine)
Counterstain: an additional dye used in a microscopy specimen to produce a contrasting background or to make clearer the distinction between different kinds of tissue. (Safranin)
3-9 Capsule Stain (differentialstain)
Figure 3-103 Example of a capsule stain
Figure 3-104 Procedure for capsule stain
Capsule: a gelatinous layer forming the outer surface of some bacterial cells. Composed of mucoid polysaccharides or polypeptides that repel most stains. The capsule remains unstained and appears as a white halo between the cells and the colored background.
3-10 Endospore Stain (differentialstain)
Figure 3-105 The Schaeffer-Fulton Spore Stain Steps
Figure 3-106 Examples of endospore stain
Figure 3-107 Example of endospore stain
Figure 3-109 Procedure of endospore stain
Endospore: environmentally resistant structure produced by the transformation of a vegetative cell of the gram-positive genera bacillus or clostridium. Is a dormant form of the bacterium that allows it to survive poor environmental conditions.
Keratin: spores are resistant to heat and chemicals because of a tough outer covering made of the protein keratin.
Vegetative Cells: any of the cells of a plant or animal except the reproductive cells; a cell that does not participate in the production of gametes; "somatic cells are produced from preexisting cells.
5-6 Oxidase Test
Figure 5-22 Oxidase Slide Test
Table 5-6 Oxidase Test Results and Interpretations
[*]Summary: The oxidase test is designed to identify the presence of cytochrome c oxidase. Transfer some of the culture to the reagent slide. Observe the color change within 20 seconds. Look at table 5-6 for results.
5-8 Citrate Test
Figure 5-30 Citrate Test Result Tubes
Table 5-8 Table of the Results
[*]Summary: the citrate test detects the ability of an organism to use citrate as the sole source of carbon and energy. A medium containing citrate as the only available carbon source, bacteria that possess citrate-permease can transport the molecules into the cell and enzymatically convert it to pyruvate. Pyruvate then can be converted to a variety of products, depending on the pH of the environment. Bacteria that survive in the medium and utilize the citrate also convert the ammonium phosphate to ammonia and ammonium hydroxide, both of which tend to alkalinize the agar. As the pH goes up, the medium changes from green to blue. Blue is a positive citrate test result. Occasionally a citrate-positive organism will grow without producing a change in the color, so you have to look for growth, which will be a positive result.
Figure 5-43 Starch Hydrolysis Test Example
Table 5-12 Starch/Amylase Test Results and Interpretations
[*]Summary: Starch is too large to pass through the bacterial cells membrane. It first must be split into smaller fragments or individual glucose molecules. Organisms that produce and secrete the extracellular enzymes a-amylase and oligo-1,6-glucosidase are able to hydrolyze starch by breaking the glycosidic linkage between sugar units. When breaking the starch, they hydrolyze the starch in the area surrounding their growth. Therefore, if there is a clearing around the bacteria, that means the amylase is present.
5-13 Urea Hydrolysis
Figure 5-46 Urease Broth Test Results
Table 5-14 Table of Results (The syllabus said to look at table 5-13. However, we only did table 5-14 in lab. Therefore, I’m assuming that this is another error).
[*]Summary: Urea is a product of decarboxylation of certain amino acids. It can be hydrolyzed to ammonia and carbon dioxide by bacteria containing the enzyme urease. Urea hydrolysis to ammonia by urease-positive organisms will overcome the buffer in the medium and change it from orange to pink. The pH indicator turns pink when media turns alkaline. Therefore, if the color turns pink, that means there is rapid urea hydrolysis. If the color is orange or yellow, there is no urea hydrolysis, because the organism does not produce urease or cannot live in broth.
5-14 CaseinHydrolysis Test
Figure 5-48 Casein Hydrolysis Test Results
Table 5-15 Casease Test Results and Interpretations
[*]Summary: Casease is an enzyme that some bacteria produce to hydrolyze the milk protein casein. When broken down into smaller fragments, the ordinarily white casein loses its opacity and becomes clear. Casease-positive organism, secrete casease will diffuse into the medium around the colonies and create a zone of clearing where the casein has been hydrolyzed. Casease-negative organisms do not secrete casease and do not produce clear zones around the growth.
5-15 Gelatin Hydrolysis Test
Figure 5-50 Nutrient Gelatin Stabs
Table 5-16 Gelatinase Test Results and Interpretations
[*]Summary: Gelatinases comprise a family of extracellular enzymes produced and secreted by some microorganisms to hydrolyze gelatin. When a tube of Nutrient Gelatin is stabinoculated with a gelatinase-positive organism, secreted gelatinase will liquefy the medium. Gelatinase-negative organisms do not secrete the enzyme and do not liquefy the medium.
5-17 Lipid Hydrolysis Test
Figure 5-55 Lipid Hydrolysis Test Results
Table 5-18 Lipase Test Results and Interpretation
[*]Summary: The enzymes that hydrolyze fats are called lipases. Some bacteria produce lipases to hydrolyze fats. Tributyrin oil is the most common constituent of lipase-testing media, because it is the simplest triglyceride found in natural fats and oils. If the bacterium produces lipase, there will be a clearing in the agar around growth. If the bacterium doesn’t produce lipase, there will be no clearing in the agar around growth.
5-20 SIM Medium
Figure 5-64 Sulfur Reduction in SIM Medium
Figure 5-67 Indole Test Results
Figure 5-68 Motility in SIM
Table 5-21Sulfer Reduction Results and Interpretations
Table 5-22 Indole Production Results and Interpretations
Table 5-23 Motility Results and Interpretations
[*]Summary: SIM medium is used for determination of three bacterial activities:
1. Sulfur reduction: Hydrogen sulfide (H2S) gas is produced. The H2S combines with iron, in the form of ferrous ammonium sulfate, to form ferric sulfide (FeS), a black precipitate. Therefore, if it is black, then the sulfur was reduced into the production of H2S. If it is not black, then the sulfur was not reduced.
2. Indole production from tryptophan: Some bacteria produce the enzyme tryptophanase, which can hydrolyze to pyruvate, ammonia, and indole. If there is red in the alcohol layer of Kovac’s reagent, then the tryptophan is broken down into indole and pyruvate. If the reagent color is unchanged, then trptophan is not broken down into indole and pyruvate. Therefore, they lack the enzyme tryptophanase.
3. Motility: The medium is inoculated with a single stab from an inoculating needle. Motile organisms are able to move about in the semisolid medium and can be detected by the radiating growth pattern extending outward in all directions from the central stab line. If you see growth radiating outward from stab line, that means there was motility. If there is no radiating growth, that means the organisms are nonmotile.
Exercise 5-12 through 5-17:
Know enzymes: amylase, urease, casease, gelatinase, lipase
Other relevant terminology from powerpoint
Incubator: an incubator is a device used to grow and maintain of course microbiological cultures or cell cultures. The incubator maintains optimal temperature, humidity and other conditions such as the carbon dioxide (CO2) and oxygen content of the atmosphere inside.
Magnification: is the process of enlarging something only in appearance, not in physical size.