Microbiology Lab

What do deamination and decarboxylation reactions have in common?

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.

In your own words, what are the roles of crystal violet and bile salts in MacConkey agar?

Both are selective agents that inhibit Gram-positive organisms.

Would removing b-phenylethyl alcohol from PEA alter the medium's sensitivity or specificity?

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.

Compare the recipes of nutrient agar and MacConkey agar. If an organism can grow on both media, on which would you expect it to grow better? Explain your answer.

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).

In your own words, what are the roles of neutral red and lactose in MacConkey agar?

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.

Which ingredients in MacConkey agar supplies Nitrogen?

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.

In your own word, what is the application (purpose) of PEA?

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.

Why is it important to apply even pressure to the plate when sampling the surface?

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.

Considering the cultures used to inoculate each medium in this exercise, how many different microbial types should you expect to see on/in each medium? Explain your answer.

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.

Examine the quadrant streak and T-streak plates. Have your lab partner write a critique of your isolation technique in the space below. The following should be addressed: Was isolation produced on one or both plates? On the quadrant streak, were the first three streaks near the edges of the plate? On both plates, did any streaks intersect streaks they shouldn't have? Was the whole surface of the agar used? Was the agar cut by the loop?

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

If you observed the same organism on a prepared slide and a wet mount, how did the images compare?

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.

What information is provided by sampling the surface immediately after treatment with disinfectant?

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

What information would be provided by sampling the surface several hours after treatment with disinfectant?

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

Phenylethyl Alcohol Agar

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.

Spread Plate Method

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.

Reservoir

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

Genus Proteus

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

Most colonies on streak plates grow from isolated colony-forming units (CFUs). On rare occasions, however, a colony can be a mixture of two different organisms. If a culture is started from this colony (thinking it is pure), correct identification will be next to impossible because the extra organism could confound the identifying test results. How could you verify the purity of a colony? (The answers may vary depending on what experience you have had prior to performing this exercise.) If you found the colony to be a mixture of organisms, what could you do to purify it?

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.

Simmons Citrate Agar

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.

How would you expect both positive and negative results to be affected if you were to add glucose to the medium?

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.

Did you achieve isolation using the quadrant streak? If so, in which streak (1, 2, 3, or 4) did it occur? If you did not achieve isolation, what might you do differently next time to improve your results?

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.

Which medium was most difficult for you to transfer from? Which medium was most difficult for you to inoculate? Explain your difficulties. (Note: There are not correct answers to these questions. They are based on evaluation of your personal experience).

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

Did you notice a difference in density of growth on NA slants inoculated from NA slants and NB? Suggest possible reasons why a difference might occur.

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

Examine the environmental sample. Did you achieve isolation?

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

Did you achieve isolation using the T-streak? If so, in which streak (1, 2, or 3) did it occur? If you did not achieve isolation, what might you do differently next time to improve your results?

Answers will vary.

Did you notice a difference in density (turbidity) of growth in NB tubes inoculated from NB and NA slants? Suggest possible reasons why a difference might occur.

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.

Were the there different streak methods appropriate to the cell densities recovered?

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.

Did you get growth on/in the sterile NB and NA slant tubes you practiced with? If not, congratulations. If so, where did you see it and what might have been its source(s)?

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.

Which medium was most difficult to prepare without contamination? Why do you think this might be so?

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.

What ingredient in PEA supplies Nitrogen?

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

Liquid media

Broth media (used for growing large numbers of bacteria)

Opportunistic pathogens

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

Which ingredient in PEA supplies Carbon?

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

Many bacteria that are able to metabolize citrate (as seen in the citric acid cycle) produce negative results in this test. Why? Be specific.

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 (!).

Descriptions of Colony Morphology

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

Nutrient Broth and Nutrient 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.)

Nutrient Agar

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

Chocolate agar

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

MacConkey Agar

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.

Thayer-Martin

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

Chemically Defined Media

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

Chemically Defined Media

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

Complex Media

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.

Complex media

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

Differential Media

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

Differential Media

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

Amies

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).

Using your pencil, perform a quadrant streak on the "practice plate" below.

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

Which ingredients in MacConkey agar supplies carbon?

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

Utilization Media

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.

Suppose a mistake is made in preparing a batch of MacConkey agar and he starting pH is 7.6 instead of 6.9-7.3. Would that affect the medium's sensitivity of specificity? Explain your answer.

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.

Why is it important that the plate be constructed so the lid is held above the agar (and not resting on it) during incubation?

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

Three critical aspects of a description of bacterial growth are colony size, color, and shape. At least three other important factors-not descriptions of the organism itself- typically are included when describing bacterial growth. What do you suppose they are and why they are important?

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.

Selective Media

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

Selective Media

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

If you are performing this test on an unknown organism, why is it a good idea to run simultaneous tests on known phenylalanine-positive and phenylalanine-negative organisms?

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.

In many tests it is acceptable to read a positive result before the incubation time is completed. Why is this not the case with starch agar?

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.

Would removing bile salts and/or crystal violet from MacConkey agar alter the medium's sensitivity and specifity? Explain your answere.

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.

Is PEA a defined or undefined medium? Provide the reasoning behind your choice and explain why this formulation is desirable

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.

Is MacConkey agar a defined or an undefined medium? Provide the reasoning behind your choice and explain why this formulation is desirable.

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.

A description of colony morphology (shape) provides important information about an organism. What other colony features should you include?

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.

In your own words, what is the role of b-phenylethyl alcohol in PEA and how does it work?

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.

Phenyalanine Deaminase

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.

Whole colony terms

Round, irregular, filamentous, and rhizoid.

MacConkey Agar (MAC)

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

Streak-plate method

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

Why is it important not to slide the plate when sampling the surface>

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

Margin terms

Smooth, rhizoid, irregular, lobate, and filamentous

What features did the cells you observed have in common? How were they different?

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

Explain how an organism that possesses the citrate lyase enzyme might not test positively on Simmons citrate agar. Is this a false negative result? Why or why not?

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.

Why is it important to counteract the effects of disinfectant with Polysorbate 8- and Lecithin during incubation?

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.

agar

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

pure culture

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

aseptic technique

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

For each of the following types of contamination suggest the most likely point in preparation (or later) at which the contaminant was introduced. a. Growth in all broth tubes. b. Growth in one broth tube. c. Growth only on the surface of the plate. d. Growth throughout the agar's thickness on a plate. e. Growth only in the upper 1 cm of agar in an agar deep tube. f. All plates in a batch have the same type and density of contaminants. g. Only a few plates in a batch are contaminated, and each looks different.

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.

Solid Media

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

lag phase

cells begin synthesizing enzymes required for growth

Blood agar

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.

Elevation terms

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

Carbohydrate fermentation

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.

Pure Culture

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

S. Marcescens

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

numerical aperture

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