MICRO Lab Practical #2 (will continue to update)

Amplification

-The act or result of increasing in size or effect. -An increase in the frequency of a gene or chromosomal region, as a result of replicating a DNA segment by in vivo or in vitro process, such as by gene duplication or polymerase chain reaction, respectively.

Least Effective Category =

Antibiotic

Understand the roles and the physical characteristics/appearance of different WBCs including:

see below

Given a plate with antibiotic disks measure the zone of inhibition using a ruler, and describe which antibiotics were most/least effective.

see below for results

Know how to leave your bench top after lab is finished

o Spray bench and leave "wet"

For each of the antibiotics tested describe its action as being directed against the cell wall, translation, DNA replication or general metabolism:

see below

For each of the metabolic tests below be able to: identify the test by site, name the purpose of the test (what is being tested), describe how this is being tested, describe any indicators used, describe the possible outcomes of the test (positive vs. negative results):

see below

In terms of the thiogylcollate test (2-6) understand the terms aerobic, anaerobic, obligate aerobes, facultative anaerobes, obligate anaerobes, aerotolerant anaerobes.

see below

basophils

-large blue/purple granules -release histamine -phagocytosis: low -diapedesis: low

Least Effective Inhibition Type

Cell wall inhibition

Most Effective Antibiotic =

Ciproflox

Be able to calculate the original phage density given a plate and dilution series problem (similar to extra credit assignment).

PFUs (plaque forming units) / (TDFx)(Vol. plated)

Most Effective Category =

Synthetic

facultative anaerobes

-A facultative anaerobe is an organism which can survive in the presence of oxygen, can use oxygen in aerobic respiration, but can also survive without oxygen via fermentation or anaerobic respiration.

granulocytes

-A leukocyte characterized by the presence of numerous staining granules in the cytoplasm when observed by light microscopy. -Neutrophils. -Eosinophils. -Basophils.

Agar slants

-Agar slants are created by bringing agar to the boiling point and pouring it into a test tube. Before the agar cools and solidifies, the test tube is set on its side. Once the agar is cooled, the test tube can be stored upright, and the agar inside has a slanted appearance. -Slanting the surface of the agar gives the bacteria a greater surface area on which to grow in a test tube. Furthermore, slants are created in test tubes that can be capped, which minimizes water loss. This is important because of the high moisture content of agar media. -Agar slants can be used to culture bacterial cells for identification. Attempting to identify bacteria from a large sample is difficult because bacteria are small and can be difficult to find. However, when placed on a nutrient agar slant, bacterial cells will divide quickly and within several hours will have produced enough cells to examine microscopically. Agar slants are also useful in maintaining bacterial cultures, more so than stacks of Petri dishes. Multiple cultures are easily placed into test tube racks and stored under refrigeration.

monocytes (macrophages and dendritic cells)

-largest cell size -U shaped nucleus -2nd responder -long lived -matures into macrophages -phagocytosis: high -diapedesis: high

neutrophils

-multi-lobed nucleus -1st responder -number increases when infection is present -phagocytosis: high -diapedesis: high

Agar deeps

-An Agar Deep Stab is a tube filled with a solid medium containing agar. It is inoculated with a needle to observe the growth patterns in the agar. Due to the presence of agar (which slows the diffusion of oxygen from the air), and the process of autoclaving and the presence of bacteria (both tend to deplete oxygen from the tube), an oxygen gradient is set up in the tube such that high concentrations of oxygen are found at the top and low to little are found at the bottom. The presence and location of growth in the tube then indicates the oxygen requirements for any given organism.

aerobic

-An aerobic organism or aerobe is an organism that can survive and grow in an oxygenated environment.

anaerobic

-An anaerobic organism or anaerobe is any organism that does not require oxygen for growth. It may react negatively or even die if free oxygen is present.

Immunocompromised

-An immunocompromised host is a patient who does not have the ability to respond normally to an infection due to an impaired or weakened immune system. This inability to fight infection can be caused by a number of conditions including illness and disease (eg, diabetes, HIV), malnutrition, and drugs.

Inoculating loop

-An inoculation loop, also called a smear loop, inoculation wand or microstreaker, is a simple tool used mainly by microbiologists to retrieve an inoculum from a culture of microorganisms. The loop is used in the cultivation of microbes on plates by transferring inoculum for streaking. It can also be used to transfer microscopic organisms. Touching a broth or a culture plate will gather enough microbes for inoculation. -The wire forms a small loop with a diameter of about 5 mm. The loop of wire at the tip may be made of platinum, tungsten or nichrome, the latter being inferior but less expensive. This loop removes a consistent amount of the liquid suspended inoculum by using the phenomenon of surface tension. -The inoculation loop is sterilized (even automatically) with flame (or another heat source) before and after each use. By doing this, the same tool can be reused in different experiments without fear of cross-contamination. After flame sterilization, the loop must be cooled so that the next cells the loop touches are not killed by the hot metal.

Inoculating needle

-An inoculation needle is a laboratory equipment used in the field of microbiology to transfer and inoculate living microorganisms. It is one of the most commonly implicated biological laboratory tools and can be disposable or re-usable. A standard reusable inoculation needle is made from nichrome or platinum wire affixed to a metallic handle. A disposable inoculation needle is often made from plastic resin. The base of the needle is dulled, resulting in a blunted end.

antibiotics

-Antibiotics are chemicals that kill or inhibit the growth of bacteria and are used to treat bacterial infections. They are produced in nature by soil bacteria and fungi.

Describe the differences between the terms: a. Antibiotic b. Semi-synthetic c. Synthetic

-Antibiotics: products of metabolism of microbes that kill or inhibit other microbes (created in nature) -Semi-synthetics: altered antibiotics (by man) -Synthetics: completely artificial antimicrobials (by man)

Broths

-Broth is a nutrient-infused liquid medium used for growing bacteria.

Describe how foods may become contaminated.

-Contamination can occur at any point along the chain—during production, processing, distribution, or preparation. -If a hen's reproductive organs are infected, the yolk of an egg can be contaminated in the hen before it is even laid. -If the fields are sprayed with contaminated water for irrigation, fruits and vegetables can be contaminated before harvest. -Fish in some tropical reefs may acquire a toxin from the smaller sea creatures they eat. -If contaminated water or ice is used to wash, pack, or chill fruits or vegetables, the contamination can spread to those items. -During the slaughter process, germs on an animal's hide that came from the intestines can get into the final meat product. -If germs contaminate surfaces used for food processing, such as a processing line or storage bins, germs can spread to foods that touch those surfaces. -If refrigerated food is left on a loading dock for long time in warm weather, it could reach temperatures that allow bacteria to grow. -Fresh produce can be contaminated if it is loaded into a truck that was not cleaned after transporting animals or animal products. -If a food worker stays on the job while sick and does not wash his or her hands carefully after using the toilet, the food worker can spread germs by touching food. -If a cook uses a cutting board or knife to cut raw chicken and then uses the same knife or cutting board without washing it to slice tomatoes for a salad, the tomatoes can be contaminated by germs from the chicken. -Contamination can occur in a refrigerator if meat juices get on items that will be eaten raw. -Sometimes, by the time a food causes illness, it has been mishandled in several ways along the food production chain. Once contamination occurs, further mishandling, such as undercooking the food or leaving it out on the counter at an unsafe temperature, can make a foodborne illness more likely. Many germs grow quickly in food held at room temperature; a tiny number can grow to a large number in just a few hours. Reheating or boiling food after it has been left at room temperature for a long time does not always make it safe because some germs produce toxins that are not destroyed by heat.

enteric

-Enteric bacteria are bacteria of the intestines, and may refer to: -Gut flora, which are always present and usually harmless -Pathogenic bacteria of bacterial gastroenteritis -The taxonomic family Enterobacteriaceae

Understand the purpose of serial dilutions. a. What is a countable plate?

-Serial dilutions are used to calculate the concentration of microorganisms. As it would usually be impossible to actually count the number of microorganisms in a sample, the sample is diluted and plated to get a reasonable number of colonies to count. Since each colony on an agar plate theoretically grew from a single microorganism, the number of colonies or Colony Forming Units is representative of the number of viable microorganisms. Since the dilution factor is known, the number of microorganisms per ml in the original sample can be calculated. -The countable plate has between 30 and 300 colonies. More than 300 colonies would be difficult to count, and less than 30 colonies is too small a sample size to present an accurate representation of the original sample. As stated above, the number of colonies is the number of Colony Forming Units which represents the number of microorganisms per ml.

Define the term bacteriophage or phage. Understand how plaques form.

-Even bacteria can get a virus! The viruses that infect bacteria are called bacteriophages, and certain bacteriophages have been studied in detail in the lab (making them some of the viruses we understand best). -Two different cycles that bacteriophages may use to infect their bacterial hosts: (1)The lytic cycle: The phage infects a bacterium, hijacks the bacterium to make lots of phages, and then kills the cell by making it explode (lyse). (2)The lysogenic cycle: The phage infects a bacterium and inserts its DNA into the bacterial chromosome, allowing the phage DNA (now called a prophage) to be copied and passed on along with the cell's own DNA. -A viral plaque is a visible structure formed within a cell culture, such as bacterial cultures within some nutrient medium (e.g. agar). The bacteriophage viruses replicate and spread, thus generating regions of cell destructions known as plaques. -Counting the number of plaques can be used as a method of virus quantification. These plaques can sometimes be detected visually using colony counters, in much the same way as bacterial colonies are counted; however, they are not always visible to the naked eye, and sometimes can only be seen through a microscope, or using techniques such as staining (e.g. neutral red for eukaryotes or giemsa for bacteria) or immunofluorescence. Special computer systems have been designed with the ability to scan samples in batches. -The appearance of the plaque depends on the host strain, virus and the conditions. Highly virulent or lytic strains give clear plaques while strains that only kill a fraction of their hosts (due to partial resistance/lysogeny) or only reduce the rate of cell growth give turbid plaques. Some partially lysogenic phages give bull's-eye plaques with spots or rings of growth in the middle of clear regions of complete lysis.

Differential media

-Ex: EMB -This is a media that is used for differentiating between bacteria by using an identification marker for a specific type of microorganism. -The selective and differential culture media are opposites to each other in a way that one inhibits the growth of other organisms while allowing the growth of some while the other does not kill the others but only highlights one type. -Blood agar is a common differential culture medium used to identify bacteria that causes haemolysis in blood.

Selective media

-Ex: MSA and Macconkey -This is a special type of media which allows the growth of certain microorganisms while inhibits the growth of the others. -For example if we want to isolate a specific bacteria let's say that can with stand an acidic environment from a sample of pond water and get rid of others, a selective media with a low pH will be taken which will allow the growth of only those organisms that can withstand acidity and will kill the others that cannot. -Examples of commonly used selective media includes: PALCAM agar medium or Mac conkey agar medium.

General purpose media

-Ex: TSA -The general purpose media is a media that has a multiple effect, i.e. it can be used as a selective, deferential or a resuscitation media.

Ciproflox

-Inhibition: DNA Inhibitor -Type: synthetic A bacteriostatic synthetic, it inhibits bacterial DNA replication.

Ofloxacin

-Inhibition: DNA Inhibitor -Type: synthetic A bacteriostatic synthetic, it inhibits DNA gyrase, inhibiting DNA replication.

Vancomycin

-Inhibition: cell wall -Type: antibiotic A bactericidal antibiotic, it inhibits proper cell wall synthesis in gram-positive bacteria.

Amoxicillin

-Inhibition: cell wall -Type: semi-synthetic A bacteriostatic semi-synthetic, it attaches to the cell wall of susceptible bacteria and results in their death by beta-lactam ring structures preventing peptidoglycan formation/re-structuring.

Ampicillin

-Inhibition: cell wall -Type: semi-synthetic A bacteriostatic semi-synthetic, it inhibits the enzyme transpeptidase, which is needed by the bacteria to make the cell wall; it can penetrate gram-negative cell membranes.

Telavancin

-Inhibition: cell wall -Type: semi-synthetic A bactericidal semi-synthetic derivative of vancomycin, it inhibits bacterial wall synthesis by binding to the peptidoglycan in the growing cell wall and disrupts bacterial membranes by depolarization.

Comp. Sulphonamides

-Inhibition: metabolism -Type: synthetic A bactericidal synthetic, it performs competitive inhibition and limits the production of folic acid.

Gentamycin

-Inhibition: translation -Type: antibiotic A bactericidal antibiotic, it inhibits protein synthesis and prevents translation by altering ribosomes.

Erythromycin

-Inhibition: translation -Type: antibiotic A bacteriostatic antibiotic (it has been synthesized), in inhibits bacterial cell wall synthesis by binding to the bacterial rRNA complex.

agranulocytes

-Leukocyte lacking apparent cytoplasmic granules when viewed under light microscopy (in contrast to granulocytes). (lymphocytes and monocytes)

obligate aerobes

-Obligate aerobes need oxygen because they cannot ferment or respire anaerobically. They gather at the top of the tube where the oxygen concentration is highest.

obligate anaerobes

-Obligate anaerobes are poisoned by oxygen, so they gather at the bottom of the tube where the oxygen concentration is lowest.

Pasteur Pipette

-Pasteur pipettes, also known as droppers or eye droppers, are used to transfer small quantities of liquids. They are usually glass tubes tapered to a narrow point, and fitted with a rubber bulb at the top. The combination of the Pasteur pipette and rubber bulb has also been referred to as a teat pipette.

Opportunistic pathogen

-Pathogens can be classified as either primary pathogens or opportunistic pathogens. A primary pathogen can cause disease in a host regardless of the host's resident microbiota or immune system. An opportunistic pathogen, by contrast, can only cause disease in situations that compromise the host's defenses, such as the body's protective barriers, immune system, or normal microbiota. Individuals susceptible to opportunistic infections include the very young, the elderly, women who are pregnant, patients undergoing chemotherapy, people with immunodeficiencies (such as acquired immunodeficiency syndrome [AIDS]), patients who are recovering from surgery, and those who have had a breach of protective barriers (such as a severe wound or burn).

zone of inhibition

-The clear region around the paper disc saturated with an antimicrobial agent on the agar surface. -The bacteria of interest is swabbed uniformly across a culture plate. Then a filter-paper disk, impregnated with the compound to be tested, is placed on the surface of the agar. The compound diffuses out from the filter paper into the agar. The concentration of the compound will be higher next to the disk, and will decrease gradually as distance from the disk increases. If the compound is effective against bacteria at a certain concentration, no colonies will grow wherever the concentration in the agar is greater than or equal to that effective concentration. This region is called the "zone of inhibition." Thus, the size of the zone of inhibition is a measure of the compound's effectiveness: the larger the clear area around the filter disk, the more effective the compound.

antimicrobial

-The discovery of antimicrobials like penicillin and tetracycline paved the way for better health for millions around the world. -With the development of antimicrobials, microorganisms have adapted and become resistant to previous antimicrobial agents. -Synthetic agents include: sulphonamides, cotrimoxazole, quinolones, anti-virals, anti-fungals, anti-cancer drugs, anti-malarials, anti-tuberculosis drugs, anti-leprotics, and anti-protozoals. -Key Terms: -antimicrobial: An agent that destroys microbes, inhibits their growth, or prevents or counteracts their pathogenic action. -microorganism: An organism that is too small to be seen by the unaided eye, especially a single-celled organism, such as a bacterium. -bacteria: A type, species, or strain of bacterium.

Normal microbiota

-The phrase "normal microbiota " refers to the microorganisms that reside on the surface and deep layers of skin, in the saliva and oral mucosa, in the conjunctiva, and in the gastrointestinal tracts of every human being. -These microbiota are not harmful to humans; some are even beneficial and most help maintain our health. -Our normal microbiota consists of various bacteria, fungi, and archaea. -While our bodies are happy to host the array of microbiota that are considered "normal," the human body does not take a back seat when infection tries to use it as a host. -Resistance to and recovery from viral infections depends on the interactions that occur between virus and host. The host has a variety of barriers that it uses to prevent infection. -One of the first lines of defense is mucus, which has a range of normal microbiota. -There are a number of other humoral components of the nonspecific immune system as well.

Serological pipette (be able to use)

-The serological pipette is a nearly ubiquitous laboratory instrument used for transferring milliliter volumes of liquid. Serological pipettes typically have gradations along their sides for measuring the amount of liquid being aspirated or dispensed. -These instruments are most commonly used with a pipette dispenser, which facilitates the liquid transfer through the creation of a partial vacuum. The same pipette dispenser can be used with a variety of serological pipette sizes, depending on the amount of volume you wish to transfer.

aerotolerant anaerobes

-Today, some bacteria species called aerotolerant anaerobes do not use oxygen at all, but they have evolved to survive in oxygen-rich environments. 'Aero' refers to oxygen, so aerotolerant means the cells can tolerate the oxygen.

leukocytes

-White blood cells -Any of the nucleated blood cells that lack hemoglobin, and whose main function involves the body's immune system, protecting the body against invading microorganisms and foreign particles.

Pure culture

-a culture in which only one strain or clone is present. -Microbial cultures are foundational and basic diagnostic methods used extensively as a research tool in molecular biology. It is often essential to isolate a pure culture of microorganisms. A pure (or axenic) culture is a population of cells or multicellular organisms growing in the absence of other species or types. A pure culture may originate from a single cell or single organism, in which case the cells are genetic clones of one another.

Agars

-a gelatinous substance obtained from various kinds of red seaweed and used in biological culture media and as a thickener in foods. -Agar is a gelatinous polymer substance derived from red algae and commonly used in a biological laboratory setting as a substrate. Agar plates are petri dishes containing agar in combination with a growth medium to culture microorganisms such as bacteria. After planting initial microorganisms on the gelatinous surface of the plate, researchers incubate them at body temperature to form colonies for isolation and analysis. Agar plates come with many different types of media or nutrients, depending on the microorganism you want to culture. -Blood agar plates (BAP) are made by adding five to ten percent sheep or horse blood to the nutrient medium. The red blood cells remain intact in the agar and make the plates a blood red color. It is an enriched, non-selective differential medium that supports growth of a variety of bacteria and can detect hemolytic activity of the microorganism. Bacteria can be beta-hemolytic and lyse the red blood cells in the agar, alpha-hemolytic and partially lyse the red blood cells, or gamma (non)-hemolytic and cause no change in the red blood cells. Chocolate agar plates (CHOC) do not actually contain chocolate, but are a type of blood agar plate in which the red blood cells have been lysed, making the agar plates dark brown in color. They detect organisms that are difficult to grow, such as Haemophilus influenzae and Neisseria gonorrhoeae. -Nutrient agar grows the largest variety of microbes, typically fungi and bacteria. It typically contains nutrients from either beef broth or yeast extract, depending on what you want to grow. There are a few types of general nutrient agar plates. Luria Bertani (LB) agar is a common nutrient agar for the general routine growth of bacteria and is not preferentially suited toward a particular microbe type. Miller's LB agar is a variety of LB containing different proportions of the same components. Trypticase Soy agar (TSA) is another general purpose medium made with casein and soybean meal and is used as initial growth medium to observe bacterial morphology or increase bacterial growth for analysis or storage. Phenylethyl alcohol agar (PEA) is selective for species of Staphylococcus and inhibits Gram-negative bacteria. -MacConkey agar plates (MAC) are a differential type medium made with bile salts and crystal violet. These additives cause the agar to only allow growth of Gram-negative bacteria, while inhibiting the growth of Gram-positive organisms. This agar can also contain lactose to distinguish between lactose-fermenting and non-lactose fermenting bacteria by forming either red (lactose-fermenting) or clear colonies. Eosin Methylene Blue (EMB) agar plates do the same thing but use two dyes, eosin and methylene blue, to differentiate between the bacteria.

Petri dish

-a shallow, circular, transparent dish with a flat lid, used for the culture of microorganisms. -Petri dishes are one of the most common equipment in laboratories. They are round shallow transparent dishes made of glass or plastic. They come with a lid that rests loosely at the top and sides. The (borosilicate) glass types are made especially to withstand sterilization methods (e.g. by autoclaving) and for reuse. Those that are made of plastic or other synthetic materials are disposable. The newest ones have metal rings beneath to allow more efficient storage, preventing slipping and falling off when kept one on top of the other.1 The name comes from a German bacteriologist, J. R. Petri. -Petri dishes are used in the laboratory primarily for cell cultures. An agar is usually used as a culture medium for growing cells, such as microbial cells. The transparent feature of the dish is essential for observing the contents without lifting the cover. It also allows exposure of the content to light. to The looseness of the cover allow the content to allow air exchange by diffusion, thus, permitting air but preventing contamination of the contents by other microorganisms.

eosinophils

-large red granules -release chemicals that aid in the destruction of parasites -phagocytosis: low -diapedesis: high

For the transformation experiment understand the following: a. Structure of pGLO b. Selection Marker c. Ori d. GFP gene e. The arabinose operon f. The role of ampicillin g. The role of arabinose

-pGLO o Man-made plasmid that contains several common attributes o All transformation plasmids contain the following -Origin of replication (so it can be copied) -Selection marker (so that it can be found) -Gene of interest -Origin of replication o pGLO contains an ORI just like that in E. coli's chromosome o Therefore when the cell replicates pGLO will be replicated as well and passed on to the daughter cells -Selection marker o Even though lab transformation is more efficient than natural transformation it is still rare. o Potentially only 1 in a million bacteria are transformed -Problem is that many bacteria that are not transformed will survive the process -Need a way to SELECT for transformants o pGLO contains a gene that codes for resistance to an antibiotic -Bla gene = codes for the production of a beta-lactamase -This enzymes hydrolyzes ampicillin -Organisms that carry pGLO will be able to grow on plates that contain ampicillin -Organisms that lack pGLO will be sensitive to ampicillin -Gene of Interest o pGLO also carries the GFP gene -GFP = green fluorescent protein -DNA sequence for GFP comes from jelly fish -It was artificially cut into pGLO and now can be made by E. coli o The GFP gene in pGLO is behind an arabinose operon!!! -Arabinose operon works exactly like the lactose operon -Arabinose genes are turned on in the presence of arabinose (a sugar) -Arabinose genes are off when sugar is absent -GFP will only be turned on when the E. coli are fed arabinose!

lymphocytes

-smallest cell size -large round nucleus -adaptive immunity -phagocytosis: low -diapedesis: high

Contamination

-the action or state of making or being made impure by polluting or poisoning. "the risk of contamination by dangerous bacteria" -Microbiological contamination refers to the non-intended or accidental introduction of microbes such as bacteria, yeast, mould, fungi, virus, prions, protozoa or their toxins and by-products. -Prominent changes for Product Contamination include: loss of viscosity and sedimentation due to depolymerisation of suspending agents, pH Changes, Gas Production, Faulty smell, shiny viscous masses etc.

Turbidity

-the quality of being cloudy, opaque, or thick with suspended matter. -"Turbidity" is a word describing how light passes through a sample of liquid as a measure of how many particles are suspended in that liquid. For example, light will pass straight through pure water, and as a result the water will appear clear. In water containing silt, sand or chemical precipitates, however, these particles will scatter incoming light, and make the water appear cloudy. Ergo, cloudy water is more turbid than clear water. -While turbidity describes the general measure of suspended particles in a liquid, it is not reserved only for water, or for visible particles. Microbiologists use turbidity as a measure of cell density within a culture sample. Microbiologists use machines called photometers and spectrophotometers that shine different types of light through culture samples to determine turbidity. The general assumption is that the higher the turbidity, the higher number of cells within the culture.

Understand the general safety rules of the lab:

1. Wash your hands with soap and water before entering or leaving the lab. 2. Wear shoes that completely cover your feet. To avoid dropped chemicals or microbial cultures splashing on your exposed feet, you cannot wear sandals or other open-toed shoes. 3. You must wear a lab coat or large shirt when working in the microbiology lab. This serves to protect your clothing from accidental contamination during spills and aids in avoiding damage to your clothes from chemicals/dyes routinely used in the lab. You must store your lab coat/shirt in the lab. Your lab coat/shirt may NOT be removed from the room during the semester. 4. Long hair must be tied back in a ponytail to avoid fire from any flames (Bunsen burner), contamination with chemicals/dyes, and/or contamination from microbial cultures. 5. Report any accidents or injuries to your instructor as soon as possible. Know the location of the fire blanket, fire extinguisher, shower and eyewash in case of emergency. 6. Do not eat, drink, apply makeup, or smoke in the lab. Avoid hand-to-mouth activities such as nail biting or chewing on the end of a pencil/pen. 7. Disinfect your lab space at the start and end of each lab period. To disinfect your area wipe down the table top thoroughly with laboratory disinfectant (Roccal) and a paper towel. When leaving the lab spray the bench area and leave the disinfectant to air dry. 8. Remove any non-essential materials from your lab work space, and keep only what you need for conducting the laboratory activity on your bench. Store coats and backpacks where they will not be in the way. 9. At the end of the lab period return all equipment to its place of origin. It is important that we maintain/improve our surroundings for other classes/students. 10. Dispose of wastes as instructed. Hazardous chemicals and microbial cultures must be properly discarded in designated containers and kill areas. I will inform you of the proper disposal procedures. DO NOT DISCARD ANY MATERIALS THAT HAVE CONTACTED MICROBIAL CULTURES OR HAZARDOUS CHEMICALS IN SINKS OR WASTEBASKETS!

Understand the use (rules) and parts of the microscope.

1. carry with 2 hands at the arm and base 2. never use coarse adjustment knob in high power 3. focus on lowest objective lens first 4. always return to lowest power and lower stage to store -Head/Body houses the optical parts in the upper part of the microscope -Base of the microscope supports the microscope and houses the illuminator -Arm connects to the base and supports the microscope head. It is also used to carry the microscope. -Eyepiece or Ocular is what you look through at the top of the microscope. Typically, standard eyepieces have a magnifying power of 10x. Optional eyepieces of varying powers are available, typically from 5x-30x. -Eyepiece Tube holds the eyepieces in place above the objective lens. Binocular microscope heads typically incorporate a diopter adjustment ring that allows for the possible inconsistencies of our eyesight in one or both eyes. The monocular (single eye usage) microscope does not need a diopter. Binocular microscopes also swivel (Interpupillary Adjustment) to allow for different distances between the eyes of different individuals. -Objective Lenses are the primary optical lenses on a microscope. They range from 4x-100x and typically, include, three, four or five on lens on most microscopes. Objectives can be forward or rear-facing. -Nosepiece houses the objectives. The objectives are exposed and are mounted on a rotating turret so that different objectives can be conveniently selected. Standard objectives include 4x, 10x, 40x and 100x although different power objectives are available. -Coarse and Fine Focus knobs are used to focus the microscope. Increasingly, they are coaxial knobs - that is to say they are built on the same axis with the fine focus knob on the outside. Coaxial focus knobs are more convenient since the viewer does not have to grope for a different knob. -Stage is where the specimen to be viewed is placed. A mechanical stage is used when working at higher magnifications where delicate movements of the specimen slide are required. -Stage Clips are used when there is no mechanical stage. The viewer is required to move the slide manually to view different sections of the specimen. -Aperture is the hole in the stage through which the base (transmitted) light reaches the stage. -Illuminator is the light source for a microscope, typically located in the base of the microscope. Most light microscopes use low voltage, halogen bulbs with continuous variable lighting control located within the base. -Condenser is used to collect and focus the light from the illuminator on to the specimen. It is located under the stage often in conjunction with an iris diaphragm. -Iris Diaphragm controls the amount of light reaching the specimen. It is located above the condenser and below the stage. Most high quality microscopes include an Abbe condenser with an iris diaphragm. Combined, they control both the focus and quantity of light applied to the specimen. -Condenser Focus Knob moves the condenser up or down to control the lighting focus on the specimen.

coliform

A sub-class of enteric bacteria that are lactose fermenters (with gas production) -Coliforms are a group of bacteria that are used as indicators of the potential presence of pathogens, viruses or parasites in a sample. These microbes are defined as a group of bacteria having specific properties including the ability to grow at 35C in the presence of bile salts and able to ferment lactose. -Coliform bacteria are divided into two types: Total & Fecal. Total Coliform are bacteria that require complex organic nutrition and grow at elevated temperatures. They are a part of the microbial flora that makes a septic field work or they may be found in environments with high biological activity such as decomposing leaf-litter. Total Coliform bacteria in a water supply suggests that the supply is at significant risk for pathogen or parasite contamination. Fecal Coliforms are bacteria and are a normal part of feces of warm-blooded animals. The presence of these bacteria would indicate an extreme risk that the water supply could become (or is) contaminated with bacterial pathogens, enteric viruses or parasites. Escherisha coli (E.coli) is one of the fecal coliform bacteria.

Given a gram stain be able to describe the shape/arrangement of bacterial cells present.

Bacillus: rod shaped Bacillus Arrangements: Diplobacilli: Two bacilli arranged side by side with each other. Streptobacilli: Bacilli arranged in chains. Coccobacillus: Oval and similar to coccus (circular shaped bacterium) Coccus: Circular Coccus Arrangements: Cocci may occur as single cells or remain attached following cell division. Those that remain attached can be classified based on cellular arrangement: -Diplococci are pairs of cocci (e.g. Streptococcus pneumoniae and Neisseria gonorrhoeae) -Streptococci are chains of cocci (e.g. Streptococcus pyogenes). -Staphylococci are irregular (grape-like) clusters of cocci (e.g. Staphylococcus aureus). -Tetrads are clusters of four cocci arranged within the same plane (e.g. Micrococcus sp.). -Sarcina is a genus of bacteria that are found in cuboidal arrangements of eight cocci (e.g. Sarcina ventriculi).

Have a general understanding of a slide showing increased WBC counts such as leukemia.

Checkout this other image as well: (copy and paste address) https://cdn-images-1.medium.com/max/1600/1*uUW8GaUu3gCJN0XIndMDTg.png

Most Effective Inhibition Type =

DNA inhibition

Least Susceptible Bacteria =

E. Aerogenes

Calculate the following given a standard plate count and dilution series (same type of problem as extra credit homework): a. Initial dilution factor of a tube b. TDF (total dilution factor) c. Original cell density of a tube

IDF = Volume In / Volume in + Volume of Dilutent TDF = adding the exponents of all of the previous IDFs OCD = (4 steps) 1) Colonies counted (CFUs) / (TDF)(Vol. Plated) 2) Write out equation 3) Solve and put into scientific notation 4) Simplify (subtract denominator's exponent from numerator's exponent) DON'T FORGET TO LABEL YOUR UNITS! REVIEW EXAMPLES FROM CLASS

Understand where items should be disposed of (kill cart, vs. bio bin, etc.)

Kill area o Autoclaving: 121 C, 15 psi, 20 minutes Kills all microbes (exception: prions) o Kill cart will be available when necessary Broths/slants/deeps = appropriate rack on the kill cart, REMOVE tape, loosen lids o Serological pipettes will have a special location o Pasteur pipettes go in broken glass container o Agar plates = Biohazard Bags at front of class, tape plates together to prevent lids from falling off

Most Susceptible Bacteria =

M. Luteus

Methyl Red (5-3)

MR-VP = Methyl Red and Voges Proskauer Test Actually determines two things Methyl Red test: o Used to detect mixed acid fermentation o Organisms that perform mixed acid fermentation will overcome the buffer in the medium and lower the pH o Methyl Red will turn red when added to the acidic medium VP test o Tests for a SPECIFIC fermentation product called 2,3-butanediol o If 2,3-butanediol is present you will also see a red color (on top of solution) Aids in the identification of Enterobacteriaceae RECIPE: o MR-VP Broth (Chemically defined) o Reagents are added after 48 growth period MR-VP Broth Experiment (Work in table) o 5 MR-VP broths per table o Innoculate E. coli E. faecalis S. typhimurium S. epidermidis Control Tube (no bacteria)

Lysine decarboxylase test (5-8)

Media is aimed to differentiate members of enterobacteriaceae o Most of these can break down amino acids by removing the carboxyl group (COOH) -Medium contains Lysine (an amino acid) that can serve as a food source -Indicator is Bromcresol purple o Purple at basic pHs above 6.8 o Orange color at neutral o Yellow at acidic pHs below 5.2 -Organisms are incubated in broth and MINERAL OVERLAY IS ADDED o This forces which type of energy production? o At the outset _______ produces acidic byproducts! -So the media turns yellow -If decarboxylase (enzyme) is present then following fermentation of glucose, the amino acid will be used as a carbon source o This produces an amino by-product that shifts the medium back to basic o Medium will go from yellow to purple if positive -Possible results: o Yellow = glucose fermenter, no decarboxylase o Orange = non-glucose fermenter o Purple = glucose fermenter and decarboxylase

Motility (5-22)

Semisolid medium which allows bacteria to "swim" away from the streak o TTC indicator is added to tubes and turns red when bacteria are present o FIRST TIME WE HAVE USED THIS INDICATOR o TTC is colorless when reduced, but turns bright red when oxidized Bacteria steal electrons and therefore change color Possible Results: o Diffuse (RED) turbidity away from stab point is a positive result for motility o No movement away from stab point (RED stab) is a negative result for motility

Thioglycollate (2-6)

Sodium thioglycollate and L-cystine reduce oxygen to water, to test for the Oxygen requirement of a bacterium o Oxygen is removed from most of the media • Red indicator Resazurin shows the presence of oxygen at the top of the tube • Produces a gradient o Aerobic in red zone (has O2) o Fully anaerobic at the bottom • Made as a deep but is fairly "loose" (.75% agar compared to normal 1.5) o Allows movement of bacteria if they have flagella • Terminology: o Aerotolerance: describes the level of oxygen in which an organism can live o Aerobic: require oxygen ! Obligate aerobes: must have oxygen ! Facultative anaerobes: organisms that don't require oxygen but do better when it is present ! Microaerophiles: require a reduced level of oxygen o Anaerobic: cannot grow with oxygen present ! Aerotolerant anaerobes: organisms that don't require oxygen, but can survive in its presence ! Obligate anaerobes: are harmed by oxygen 1) E. Aero: a. Location: throughout b. Aerotolerance: Facultative c. Mobility: + 2) A. Faecalis: a. Location: on top b. Aerotolerance: Obligate c. Mobility: + 3) E. Coli: a. Location: throughout b. Aerotolerance: Facultative c. Mobility: + 4) S. Epi: a. Location: throughout b. Aerotolerance: Facultative c. Mobility: +

Generally describe the pour-plate technique which requires soft agar, and an agar overlay used in the bacteriophage experiment.

Soft agar is poured over hard agar on a plate with the phage and E. coli host mix. It is important to use hard agar with soft agar overlay because the hard agar underneath the soft agar overlay is where you make a lawn streak of your bacteria. Since phages can only grow in the presence of bacteria, this is the only way you can visualize plaques. When the phages lyse the bacteria, you'll see a zone of inhibition on the plate where the phages in the soft agar inhibit the bacteria on the hard agar form growing. The hard agar is necessary to suspend the soft virus bacteria agar mixture. If we did a soft agar on top of another soft agar layer, the two layers would mix. This would make it hard to count, if not impossible. The relative concentration of the phages and bacteria would no longer be known. Placing a soft agar on top of a hard agar ensures only one layer is growing and one area will need interpretation.

Least Effective Antibiotic =

Telavan

Starch hydrolysis (5-11)

Tests for the ability to hydrolyze starch o Hydrolyze = hydrolysis = break into monomeric glucose molecules (Starch is too large of a polysaccharide (carbohydrate to pass through plasma membrane) o To consume starch an organism must secrete o Amylase = enzyme that breaks starch into glucose monomers o And oligo-1,6 glucosidase (also aids starch breakdown) Bacteria are S-streaked on a plate and allowed to grow for 24 hours Iodine is used as an indicator (BIO 15 HERE WE COME) o Iodine is dripped onto the surface of the plate o Positive Result = Area around streaked out colonies will not stain (no starch available) o Negative Result = area around colonies will stain black Starch Agar Plate Experiment (Individual) o Light smears of E. coli B. cereus

Phenol red broth test (5-2)

Tests for: Bacteria are identified based largely on what organic compounds they can break down. The range of compounds used depends on the collection of enzymes a species of bacteria can make. Lactose is a sugar that some bacteria can use because of an enzyme that begins the breakdown of this compound. Phenol red lactose broth is thus used to determine whether the microbe can use the sugar lactose for carbon and energy. How is lactose fermentation determined? If lactose can be used, the microbe will accumulate acidic byproducts. In a positive test, the pH indicator in the medium changes color from its normal red to yellow, indicating acid production. What is the content of this medium? The medium is a nutrient broth to which 0.5-1.0% lactose is added. The pH indicator phenol red is red at neutral pH but turns yellow at pH <6.8. It also changes to magenta or hot pink at pH >8.4. Used to test ability to ferment a sugar and measures gas production Fermentation will produce an acidic by product Tubes contain an inverted Durham tube that will trap gas if it is produced during fermentation RECIPE: o PR Broth (Complex) o Indicator: Phenol red (same as MSA plates) red (neutral) = neg. across the board yellow (acidic) = ferm. pos. , gas pos. , deaminase? pink (basic) = ferm. neg. , gas neg. , deaminase pos. Primary outcome is to test for acidic fermentation products and gas production o Yellowing of media is + for acid production Also contains proteins that can be used as a food source, if this occurs the proteins are DEAMINATED (lose the amino group) o Produces an amino by-production o Basic o Will turn the media PINK o Only occurs if sugars are not consumed (or are exhausted) Multiple outcomes are possible, you must know ALL!!!

Catalase test (5-4)

Tests for: The catalase test is used to differentiate staphylococci (catalase-positive) from streptococci (catalase-negative). The enzyme, catalase, is produced by bacteria that respire using oxygen, and protects them from the toxic by-products of oxygen metabolism. Catalase-positive bacteria include strict aerobes as well as facultative anaerobes, although they all have the ability to respire using oxygen as a terminal electron acceptor. Catalase-negative bacteria may be anaerobes, or they may be facultative anaerobes that only ferment and do not respire using oxygen as a terminal electron acceptor (ie. Streptococci). The presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into hydrogen peroxide, and the rapid elaboration of oxygen bubbles occurs. The lack of catalase is evident by a lack of or weak bubble production. ETC contains a molecule called flavoprotein o Flavoprotein can bypass the next carrier in the ETC and pass electrons directly to oxygen (this is bad) o Produces hydrogen peroxide and superoxide radical o Organism must have enzymes to break these toxins down are it can potentially kill itself Catalase converts hydrogen peroxide into water and gaseous oxygen Slide test o 1 drop of hydrogen peroxide is added and they are mixed with a loop o Large amount of bacteria are smeared on a microscope slide o Bubbles = catalase positive o No bubbles = catalase negative Often differentiates between micrococcaceae (positive) and streptococcaceae (negative)

Glucose oxidation fermentation test (with oil) (5-1)

Tests for: The oxidative-fermentative test determines if certain gram-negative rods metabolize glucose by fermentation or aerobic respiration (oxidatively). During the anaerobic process of fermentation, pyruvate is converted to a variety of mixed acids depending on the type of fermentation. The high concentration of acid produced during fermentation will turn the bromthymol blue indicator in OF media from green to yellow in the presence or absence of oxygen . Acid production is detected in the medium by the appearance of a yellow color. In the case of oxidative organisms; color production may be first noted near the surface of the medium. Tests whether organisms can perform cellular respiration or fermentation on a specific sugar If Cellular respiration is performed then a final electron acceptor is used (oxygen) If fermentation is performed an acidic byproduct is created (lowers pH of solution) Low agar (Semi-solid motility media, like Thioglycollate tubes) Tubes are boiled to remove Oxygen o THEN ONE OF EACH PAIR IS "CAPPED WITH MINERAL OIL" = OVERLAY RECIPE: o OF Medium (Complex) o Indicator: Bromothymol blue green when neutral yellow when acidic blue when basic o Substrate: Whatever sugar you are testing Multiple outcomes are possible, you must know ALL!!!

Kligler Iron agar (5-18)

Tests for: used for the differentiation of microorganisms on the basis of dextrose and lactose fermentation and hydrogen sulfide production in a laboratory setting. Kligler Iron Agar is not intended for use in the diagnosis of disease or other conditions in humans. 1) Glucose = read the butt of the deep 2) Lactose = read the slant 3) Amino acid consumption = read slant, only can see if lactose is negative 4) Gas = cracks or splits in the agar 5) H2S = black precipitate (only occurs if glucose is positive as well) Medium tests several bacterial attributes: o Ability to ferment glucose o Ability to ferment lactose o Ability to reduce sulfur (use this as a final electron acceptor!) Unique to our tests in that it is made as a "deep slant" o Inoculated with stab and streak method Indicators present: o pH indicator is phenol red (will turn ????) o Ferrous sulfate acts as a hydrogen sulfide indicator (turns black when hydrogen sulfide is present) Potential food sources available o Glucose o Lactose o Peptone (amino acids) POSSIBLE RESULTS (DIFFICULT) o Glucose fermentation only: tube will yellow completely, then after glucose is exhausted the slant (top) will revert to red/pink due to amino acid consumption o Glucose and lactose fermentation: tube will yellow completely (high lactose concentration is not exhausted so more acid is produced) o Gas production during fermentation leads to "cracked" or raised agar o No fermentation: amino acids are used, alkaline products turn the medium red/pink (if peptone used) o Use of sulfur as an electron acceptor produces H2S H2S reacts with ferrous sulfate to form black precipitate (only in presence of ACID)

Citrate test (5-7)

Tests for: utilization of citrate as the sole carbon source, nitrogen consumption and processing turns media alkaline if positive (ANY growth is positive) Color Changes: basic (blue = positive) neutral (green = negative) -A type of utilization media: o Specifically tests to see if the organism can utilize an essential nutrient o Limits the bacterial food sources -Bacteria are provided citrate o The first product of the Kreb's cycle Oxaloacetate combines with AcetylCoA to form Citrate o Citrate is the only carbon source provided If the bacteria contain citrate permease o They can transport citrate into the cell (it becomes permeable) o Once in the cell it can be converted back to pyruvate o Pyruvate can be used to create a variety of products -Bacteria that use citrate also must have a nitrogen source o Use materials in the media to create ammonia o Makes the media more alkaline (basic) -Indicator is bromthymol blue (turns blue when basic) o Positive test is converting the tube from green to blue o Same indicator used in the OF tests!!! Substrate: Citrate Indicator: Bromthymol blue INNOCULATED WITH A NEEDLE TO AVOID HEAVY INNOCULATION (DO NOT STAB)!!!

Understand the general concept of the food experiment with supermarket samples including dilution series, and coliforms vs. non-coliforms.

The general concept is to test several food sources from the grocery store for contamination. It also helps to understand whether enterics are present in food or water is critical during epidemics that show characteristics of enteric caused diseases. Dilution series: Coliforms vs. non-coliforms: Enterics (non-coliform) are gram-negative, facultative anaerobes that have evolved to live in the intestines of vertebrates; Enterics can survive in the wild as decomposers and so alone are not "sure" indicators of fecal contamination; some are pathogenic. Coliforms are a cub-class of enteric bacteria that are lactose fermenters (with gas production); they are much more likely to indicate fecal contamination of food and water sources; most are non-pathogenic (exist commensally). Enterics and coliforms are shed in feces of animals and humans; the presence of coliforms (especially E. coli) is an indication of fecal contamination; it doesn't confirm that the source of the disease is in the sample, but it is a strong indicator that the sample may carry harmful non-coliforms (salmonella, shigella, etc.)

Understand the general purpose of the following media types during the food experiment: a. Lauryl Tryptose Broth (LTB) b. Brilliant Green Lactose Bile Broth (BGLB) c. EC w/MUG broth

a.Lauryl Tryptose Broth (LTB): Generally screens for enterics and coliforms; it does allow other bacteria to grow (growth=contamination); gas production is visualized with a Durham tube (coliform specific) b. Brilliant Green Lactose Bile Broth (BGLB): Allows for the growth of enteric bacteria; bile inhibits the growth of non-enterics; gas production (coliform specific) if visualized with a Durham tube; positive indicator that enterics are present. c. EC w/MUG broth: Most E. coli strains contain the enzyme B-glucuronidase; MUG is a synthetic chemical that when cut will fluoresce; 360 nm UV light is used to determine positive or negative.

For each of the above tests describe any special techniques used for inoculation, and any special materials used (Durham tubes for instance).

b. Glucose oxidation fermentation test (with oil) (5-1): Use a needle to stab and mineral overlays on some c. Phenol red broth test (5-2): Uses an inverted Durham tube to trap gas if it is used during fermentation h. Citrate test (5-7): Inoculated with a needle, without stabbing, to avoid heavy inoculation i. Lysine decarboxylase test (5-8): Uses a mineral overlay j. Starch hydrolysis (5-11): Uses H2O2 to create a "halo" if test is positive on an agar plate l. Kligler Iron agar (5-18): Inoculated with a stab and streak method with a needle m. Motility (5-22): Inoculated with a needle and stabbed

For MSA (4-1), EMB (4-4) and MacConkey (4-6) agar plates describe or define the following characteristics: a. Is the plate selective? If so why? b. Is the plate differential? If so why? c. What do different color outcomes tell you about the bacteria? d. Given the characteristics of a bacterium predict what color the colony or plate would turn. e. Name any bacteria that have a unique outcome.

• Selective and differential media are used to allow gram negative growth and inhibit gram positive growth • MSA: o Basic = Pink (Amino Acid Consumption) o Neutral = Red (no growth) o Acidic = Yellow (manitol fermentation positive) • Macconkey: o Yellow (basic) = lac. fermentation negative o Pink (acidic) = lac. fermentation positive • EMB: o Basic = Purple/blue o Acidic = Pink = Coliform o Very acidic = Metallic green = E. Coli a. Is the plate selective? If so why? 1. For MSA (Mannitol Salt Agar): Yes. Because with a high pH, the sodium chloride concentration inhibits the growth of most organisms. 2. For EMB (Eosin Methylene Blue) Agar: Yes. Because EMB contains dyes (Eosin Y and Methylene Blue) that are toxic to gram-positive bacteria, it is a selective stain for gram-negative bacteria (enteric bacteria/gut flora). 3. For MacConkey agar: Yes. Because it is designed to isolate gram-negative and enteric bacilli. b. Is the plate differential? If so why? 1. For MSA (Mannitol Salt Agar): Yes. Because of the presence of the sugar mannitol and the pH indicator Phenol Red, it differentiates between S. aureus and other Staphylococcus species. 2. For EMB (Eosin Methylene Blue) Agar: Yes. Because of the presence of the EMB and the sugars lactose and sucrose in the medium, it is a differential as is encourages some bacteria to grow while inhibiting others. 3. For MacConkey agar: Yes. Due to the lactose, it differentiates enterics based on their ability to ferment lactose. c. What do different color outcomes tell you about the bacteria? 1. For MSA (Mannitol Salt Agar): Yellow is below a pH of 6.8 and is acidic; Red is from a pH of 7.4-8.4 and is neutral; Pink is above a pH of 8.4. 2. For EMB (Eosin Methylene Blue) Agar: Metallic green is very acidic (E.coli) Pink/dark purple is acidic (coliform) Purple/blue is neutral (non-coliform) 3. For MacConkey agar: Yellow is basic, lactose fermentation negative Red is neutral Pink is acidic, lactose fermentation positive d. Given the characteristics of a bacterium predict what color the colony or plate would turn. 1. For MSA (Mannitol Salt Agar): Red, pink, yellow 2. For EMB (Eosin Methylene Blue) Agar: Purple, blue/purple, metallic green, pink/purple 3. For MacConkey agar: Violet, yellowish, pink e. Name any bacteria that have a unique outcome. 1. For MSA (Mannitol Salt Agar): E. coli, A. faecalis, S. aureus 2. For EMB (Eosin Methylene Blue) Agar: E. coli, E. aero 3. For MacConkey agar: E. coli, E. aero