Microbiology Lab

Contains a variety of ingredients and there is no exact chemical formula for ingredients (can be highly variable). Examples include: Nutrient Broth, Blood agar, and chocolate agar.

Composed of precise amounts of pure chemical and generally not practical for routine laboratory use (invaluable in research)

Inhibit the growth of unwanted organisms (allow only sought after organisms to grow). Example: Thayer-Martin agar MacConkey Agar

Contains substance that bacteria change in a recognizable way. Example: Blood agar MacConkey Agar

Commonly used to maintain bacterial cultures. Formulated from sources that supply C & N from a variety of sources (usually digests of plant or animal material and are considered undefined since amounts of C and N are unknown.)

Any answer that is reasonable is okay. Most students find preparing plated media aseptically most difficult. With tubed media, the medium is dispensed into the container prior to sterilization, so any contamination is removed during autoclaving. The medium has been sterilized when it is dispensed to a plate, so contaminants cannot be removed later.

a. Since the broth is sterilized after it is dispensed into the container prior to sterilization, so any contamination is removed during autoclaving. The medium has already been sterilized when it is dispensed to a plate, so contaminants cannot be removed later. b. Contamination probably occurred during storage, since the broth is sterilized after it is dispensed into the tube. c. Contamination probably occurred during storage, since it is the only on one plate and the agar had apparently solidified prior to contamination. d. Contamination probably occurred while dispensing stock medium onto the place because is is found in all levels of medium (indicating the agar was liquid when contaminated). It is not likely due to contamination of the stock medium because only one plate shows contamination. e. Contamination probably occurred as the tube was cooling after being autoclaved and prior to the agar solidifying since growth is found into the agar. f. The stock medium was probably contaminated since all plates have the same type of growth. g. Each plate was contaminated independently (probably during storage) since the contaminants differ and only a few are affected.

Answers will vary. Possibilities for contamination include not heating the loop to orange hot, not holding the open tubes on an angle, and/or place the cap on the table surface during the process.

The slant and broth cultures of S. epidermis should be pure, meaning that only S. epidermis is growing in them. Making transfers from each should result in only one microbial type on/in the receiving medium. Colonies usually arise from single or joined cells in the same species. So, transfers made from a colony should also result in one microbial type on/in the receiving medium.

Answers will vary, but generally any transfer involving holding a cap will be more difficult until the pinky finger is trained to cooperate.

Answers will vary. Generally, more cells are transferred from growth on a solid medium than from a broth culture. Therefore, broth cultures made from growth on a solid medium will show greater turbidity than those inoculated from a broth culture.

Answers will vary, but generally there will be denser growth on the slant inoculated from the NA slant for the reason stated earlier.

Examine patterns for the proper intersections and covering the whole surface.

The student evaluation should address the points raised in the question.

Answers will vary depending on technique and density of the cells in the mixture. Some possible adjustments for subsequent streak plates include: Flaming the loop between streaks. Streaking each quadrant numerous times (See Figure 1.35, p.47) rather than just a few. Using the entire agar surface rather than gravitating toward the center.

Answers will vary.

Answers will vary, but will either be "yes" or "no."

Answers will vary. In general, T-streaks and quadrant streaks are used for samples with expected high cell densities, which the S. epidermis and M. luteus cultures should have had, and therefore should have been appropriate. The zigzag streak is used for samples with expected low cell densities, so its appropriateness will depend entirely on the source each student sampled.

A Gram stain or another streak plate can be used to verify colony purity. If the colony is not pure, then it should be re-streaked (or, the streak plate used to verify purity could be used) and a colony of the "correct" organism should be picked from this second plate and used to start a pure culture.

Morphology is shape. Other features such as color and optical characteristics (shiny, opaque, etc.) are also necessary and are usually lumped in with morphology. In addition, growth medium, incubation time and temperature, and sample source should be included.

Incubation temperature, time, and the type of medium used. Temperature is important because it affects growth rate, and colonies can change in appearance as they get older. This is also why incubation time is important. Not all media contain the same resources. An organism may be able to grow on one medium, but poorly, whereas on a different medium it might grow really well. Resource availability can affect colony morphology.

Some features in common: nuclei, cytoplasm, and cell membranes. Some different features: cell walls, chloroplasts, and cilia/flagella.

There may gave been a difference in size between wet mounts and prepared slides. Also, if the prepared slide was stained differently than the wet mount, different features may have been more prominent.

PEA is a selective medium used to isolate Gram-positive organisms by inhibiting Gram negatives. It is generally used to isolate members of the genera Staphylococcus, Streptococcus, Lactococcus, and Enterococcus and inhibit common Gram negatives, such as Escherichia coli and Proteus species.

Casein (milk protein) and soybean meal provide carbon, largely in the form of the protein.

Because they contain protein, casein, and soybean act as nitrogen sources.

It is undefined due to the casein and soybean in the medium. Casein is really a related family of proteins, not one specific molecule found in the milk of all mammals so its exact composition is unknown, and soybean is a mixture of unknown biochemicals. Either one of these would make the medium undefined. The goal of this medium is to isolate desired organisms, and once the undesired organisms have been inhibited you want the desired ones to grow well. The variety of biochemicals in soybean make the medium more likely to satisfy the varied nutritional needs of the organisms being selected for.

Phenylethyl alcohol is the selective agent in PEA. It inhibits the growth of Gram-negative organisms by disrupting the cell membrane, which affects osmotic and pH homeostasis. The subsequent loss of critical molecules associated with DNA synthesis affects the cell's ability to perform DNA replication.

It would alter specificity because organisms that "shouldn't" grow on it would. It wouldn't likely alter sensitivity because you would probably still be able to detect growth of organisms that "should" grow on it.

Gelatin, casein (milk protein), animal tissue, lactose, and to some extent, bile salts are potential sources of carbon.

Organic nitrogen is mostly found in proteins and nucleic acids. Animal tissue undoubtedly has both, and casein and gelatin are both proteins, so these can act as nitrogen sources. In addition, some fatty components of bile have nitrogen.

It is undefined due to the pancreatic digests of gelatin and casein, peptic digest of animal tissue, and bile salts in the medium. An undefined medium is desirable to support growth of a wide variety of the organisms being selected for.

Both are selective agents that inhibit Gram-positive organisms.

MacConkey agar is differential medium based on the ability of an organism to ferment lactose to acid end products. Coliforms can do this, noncoliforms cannot. So, lactose is the substrate of the fermentation and neutral red is the pH indicator that detects acid production, if any.

It changes the specificity (selecting for certain Gram-positive cocci instead of Gram-negative rods). It probably wouldn't affect sensitivity, because you would still be able to detect growth of organisms that "should" grow on it as well as detect the pink color indicative of lactose fermentation.

If a batch of MAC were made were made at a higher than normal pH, it would take more acid production from lactose fermentation to produce the pink color of a positive. It is possible then, that a true positive for lactose fermentation might not change the medium's color in the allotted incubation time and would be incorrectly recorded as a negative. This would affect the medium's sensitivity.

It would probably grow better on the MacConkey agar, since there is a greater variety of carbon sources and other nutrients (pancreatic digests of gelatin and casein, peptic digest of animal tissue, and lactose) than in NA (beef extract and peptone).

Citrate (citric acid) is the first intermediate of the citric acid (Krebs) cycle where it is ultimately catabolized to CO2 and oxaloacetic acid (which re-enters the cycle). However, the citrate test does not detect the ability of an organism to perform the citric acid cycle. It detects the ability of the organism to obtain citrate from the environment and use it. Thus, an organism could synthesize its own citrate in the citric acid cycle, but not be able to use citrate from the envrionment because it lacks the ability to transport it into the cell; it is citrate permease (!).

The citrate test determines if an organism has the ability to transport citrate into the cell with the enzyme citrate permease and survive with citrate as the sole carbon source. Citrate lyase, which catalyzes the conversion of citrate into oxaloacetate and acetate, is associated with how citrate is used, not how it gets into the cell. So, an organism could be citrate-negative (it doesn't make citrate permease), but still be citrate lyase-positive.

Inoculation of a positive control (an a successful result from it) adds confidence to negative results on an unknown organism. That is, you know the test is working correctly, so the negative result is probably accurate. In the absence of the positive control, there is always an element of doubt whether the negative result is a true negative or a false negative. Performing the test on a known phenlalanine-negative organism is useful in that it demonstrates what a negative result looks like.

Both are catabolic reactions and have amino acids as substrates. They just remove different functional groups from the amino acid---deamination removes the amine and decarboxylation removes the carboxylic acid.

Addition of glucose might have the effect of slowing starch hydrolysis by amylase-positive organisms, thus creating false negatives due to the preferential use of the sugar (since glucose is a more accessible resource than starch). There would be no difference in the results shown by true amylase-negative organisms. Clearing comes only from starch digestion, not utilization of other resources in the medium.

Iodine is toxic to cells, so addition of it to the medium early ends the test. If you get lucky and a positive result is seen, then it can be recorded. If you are unlucky and a negative result is seen, it is too early to record and you have terminated the test without a usable result.

This informs the user about the immediate effectiveness of their disinfection technique and the disinfectant itself.

This informs the user about the longer-term effects of disinfection. That is, it tells how long before the disinfectant's effectiveness wears off.

The main reason is so that the sample is collected evenly across the whole surface. This is a quantitative test in that it gives the number of CFU per cm2. If the whole surface of the plate is not used, the estimate of cell density will be lower than the actual density.

Sliding the plate over the surface would mean it has contacted more than the 25 cm2 area it is intended to sample.

If the lid contacts the agar, it might interfere with colony formation, which would compromise the cell density estimate derived from that sample.

The main use of the RODAC plate is to evaluate the efficacy of disinfection of a particular surface. During sampling, contact with surface not only picks up contaminants, it also picks up disinfectant. If the disinfectant is not neutralized, cells that would grow on the plate (which are evidence of poor disinfection) might not grow because of the longer disinfectant exposure while on the plate. This would lead to a low estimate of contamination.

a set of procedures that minimize the chance of other organisms being accidentally introduced.

a population descended from a single cell and therefore contains only one species

a polysaccharide extracted from marine algae is used to solidify culture media

cells begin synthesizing enzymes required for growth

the measure of a lens's ability to "capture" light coming from the specimen and use it to make the image

population of cells derived from single cell (all genetically identical)

Broth media (used for growing large numbers of bacteria)

broth media with addition of agar, remains solid at room temperature and body temperature.

Simplest and most commonly used in bacterial isolation. Object is to reduce number of cells being spread.

Defined medium with salts to buffer osmotic pressure. Phosphate buffers for pH. Thiogycolate produces reduced environment to minimize oxidative damage. Charcoal may be present to neutralize fatty acids and bacterial toxins. No carbon of nitrogen source (for maintenance not growth).

Used to isolate microorganisms by spreading a diluted sample uniformly across the surface (uses a glass rod or metal spreader to distribute organisms). If properly diluted, CFUs will be far enough apart on surface to grow individual colonies.

complex medium used routinely in clinical labs. Differential because colonies of hemolytic organisms are surrounded by a zone of clearing of the red blood cells. Not selective.

Complex medium used to culture fastidious bacteria, particularly those found in clinical specimens. Not selective or differential.

Complex medium used to isolate Gram-negative rods that typically reside in the intestine. Selective because bile salts and dyes inhibit Gram-positive organisms and Gram-negative cocci. Differential because the pH indicator turns pink-red when the sugar in the medium, lactose, is fermented.

Complex medium used for routine laboratory work. Supports the growth of a variety of nonfastidious bacteria. Not selective or differential.

Complex medium used to isolate Neisseria species, which are fastidious. Selective because it contains antibiotics that inhibit most organisms except Neisseria species. Not differential.

Contains a variety of ingredients, there is not exact chemical formula for ingredients (can be highly variable). Ex. -Nutrient Broth -Blood agar -Chocolate Agar

Composed of precise amounts of pure chemical, generally not practical for routine laboratory use.

Inhibit the growth of unwanted organisms (allow only sought after organisms to grow) Ex. -Thayer-Martin -MacConkey Agar

Contains substance that bacteria change in recognizable way. Ex. -Blood Agar -MacConkey Agar

Capable of producing a diseased state if introduced into a suitable part of the body

an area where the microbe resides, including sites outside of the host, that can serve as a source of infection.

convex, umbonate, plateau, flat, raise, raised spreading edge, flat raised margin and growth into medium.

Smooth, rhizoid, irregular, lobate, and filamentous

Round, irregular, filamentous, and rhizoid.

Color, Surface Characteristics (Dull, shiny), Consistency (Dry, buttery, Moist), Optical properties (Opaque, Transluscent).

will swarm at certain intervals and produce a pattern of concentric rings because of their motility.

produces less orange pigment when grown at 37 degrees than when when grown at 25 degrees Celsius.

Selective and Differential. Used to isolate and differentiate members of Enterobacteriacea. Differentiates lactose fermenters from non-fermenters.

Undefined selective, inhibits Gram-negative organisms. Allows growth of Gram positive. Interferes with DNA synthesis in Gram negative (breaks down membrane permeability barrier causing leakage of cellular potassium and influx of other substances.) Used to isolate staphylococci (including enterococci and lactococci). Typically used to screen out common contaminants E. coli and Proteus species.

metabolic process by which an organic molecule acts as an electron donor (becomes oxidized) and one or more of its organic products acts as final electron acceptor.

Highly defined media, differentiate organisms on ability to grow when an essential nutrient (Carbon or nitrogen) is strictly limited, only organisms with appropriate enzymes will survive. Ex. Simmons Citrate Medium.

A defined medium, bromthymol blue dye is indicator (Green at pH 6.9 and Blue pH 7.6). Bacteria that use citrate convert ammonium phosphate to NH3 and NH4OH which alkalinizes medium.

Removes amino group (NH2) from Phe. Reaction splits a water molecule and produces NH2 and phenylpyruvic acid. PheDA activity is determined by detecting presence of phenylpyruvic acid.