Microbiology Lab Exam

#bacteria/gram or mL

# colonies x (1/dilution factor) x (1/ volume plated mL) x amount of total grams or mLs Example: 200 x (1/10^-3) x (1/.1) X 3 = 6,000,000

Final Concentration of an Enzyme

(Given concentration(ug/mL) x amount of enzyme (mL) added) / total volume (mL)

Why does gas collect in the inverted Durham tubes in the water analysis test?

At 35-37C, coliforms can ferment lactose which produces acid and gas. A positive test for coliforms is indicated by growth of bacteria in this lactose medium and collection of gas in the inverted vial. Coliforms use lactose as a carbon source while many bacteria cannot

Reservoir

Continual source of an infection

What is the purpose of covering the culture with mineral oil?

Covering the culture with mineral oil prevents oxygen from getting in. Creates an anaerobic environmental condition.

E.coli and Shigella colonies look different on EMB plates. What is responsible for this different phenotype?

Shigella colonies are colorless and have a transparent amber color because they do not ferment lactose and/or surcose. E.coli colonies may show a green metallic sheen due to rapid fermentation of lactose

Carriers

Humans who harbor pathogens but who do not exhibit any signs of disease

Why is a standard plate count performed on food products?

It is used to determine the starting concentration of bacteria in original food and water sample. It is used over other enumeration methods because it only counts living cells. It is used to analyze microbial contamination through quantitative enumeration of the microorganisms in the product.

All of the following are true about agar except: A. liquefies at about 100C B. is a polysaccharide derived from a red alga C. solidifies at approximately 40C D. is a great food source for many bacteria

Answer: D

Questions on biofilm?

-Aggregates of microbes that form layers, sheath of microbes that are usually the same species -Form where nutrients are available

Decolorizer

-Alcohol -Dissolves outer membrane of gram (-) bacteria -Allows crystal violet-iodine complex to leak out of gram (-) bacteria

Primary stain

-Crystal violet -Binds to peptidoglycan layer in both gram positive (thick) and gram negative (thin) -Purple stain

Why is the difference between food poisoning and food intoxication?

-Food poisoning: ingestion of food containing live bacteria which grow in the human intestinal tract -Food intoxication: ingestion of food containing toxins made by the bacteria

Mordant

-Iodine -Chemical that fixes in place a dye already present -Forms a complex with crystal violet in peptidoglycan layer -In gram (+), this helps resist decolorization

Counter-stain

-Safranin -Binds to thin peptidoglycan later of gram (-) -Stains gram (-) cells pink/red

What is the difference between simple and differential stains?

-Simples stains are used to give color to transparent microbial cells in order to visualize them microscopically; use only one stain -Differential stains are used to differentiate between the types of microbial cells (gram + vs -); uses 2 stains

List two possible sources of contamination that you should be concerned about while transferring bacteria from one source to another.

1. Hands 2. Touching the instruments used in the transfer to a surface (i.e laying sterile loop on a lab bench)

For some people, the "post hand washing" plate may have more colonies than the "pre hand washing." Why could this happen?

1. If the person picked microbes by touching the faucet to turn the sink off. 2. Microbes from the air getting on wet hands (bacteria enjoy moist environments)

Give three reasons for using aseptic technique

1. Minimize the potential for contamination of the culture 2. Minimize contamination of the person with microbes 3. Minimize contamination of the objects/instruments used

Why would a needle or a loop be used instead of a cotton swab to isolate individual colonies from a streak plate?

1. Needles and loops can be frequently sterilized (via flames) to avoid contamination during the process of isolation without having to be discarded after each streak. 2. Cotton swabs are used to collect bacteria from the environment and are used to place this sample onto a plate. Cannot be sterilized in the same manner, and are discarded after each use. 3. Microbes can become embedded within the cotton swab, and therefore less will be placed onto the plate.

Procedure of Gram Stain

1. Primary stain- 1 minute 2. Mordant- 1 minute 3. Decolorizer- 10-15 seconds 4. Counter-stain- 1 minute

List 2 factors that must be carefully controlled in the Kirby-Bauer disk susceptibility test method?

1. Rates of diffusion of antibiotic 2. Inoculum size

List 3 pathogens the API 20E system can be used to detect in a clinical laboratory?

1. Shigella dystentriae 2. Escherichia coli 0157 3. Klebsiella pneumoniae

Writing bacterial names

1. When you cannot italicize, underline the name 2. Genus name always begins with a capital letter, species name does not 3. If talking about a general genus (no specific species) you write spp. (underlined or italicized)

List some advantages of using solid media over broth media for the initial isolation of bacteria.

1. You can select for growth of particular bacteria by using selective plates, enriched plates, etc. 2. With solid media, you can use the streaking technique to isolate single, pure colonies of the desired bacteria. 3. Once you obtain a single colony on solid media you can transfer the colony (using sterile technique) to the broth media where it can proliferate

What temperature would be optimal to incubate an agar plate with bacteria cultured from within our bodies (e.g the gut flora)?

37C (internal body temperature)

List the final (compounded) magnifications, achieved with each objective on your microscope

40x, 100x, 400x, 1000x

What is a colony?

A group of about 10^8 cells, all of which are derived from a single parent.

What is aseptic technique?

A set of practices that are used to prevent contamination of a person, object, or an area by microbes. Prevents contamination of the specimen.

What is the benefit of using immersion oil when viewing bacteria?

Allows an increase in resolution of an object by refracting more light into the objective (and thus your eyes). Allows for a sharper image with more light. Without the oil, the light would scatter in different directions & not into the objective

Why might it be a good idea to treat with both Ampicillin and Sulbactam?

Ampicillin is a penicillin-derived antibiotic and sulbactam is an inhibitor of beta-lactamase. Both are used to treat infections of bacteria that are ampicillin resistant. Beta-lactamase breaks down ampicillin. Sulbactam blocks B-lactamase, which allows ampicillin to function and kill the bacteria

How does an autoclave work?

An autoclave is used to sterilize equipment and liquids by using high pressure steam at 121C for 15-20 minutes (varies). This high pressure steam, at a higher than boiling point temperature is more effective at killing microbes than just regular hot water.

Why are basic dyes more effective for bacterial staining than acidic dyes?

Bacterial cell walls have a negative charge due to the phospholipids and LPS (in gram -) and because of acidic polysaccharides (gram +). The (+) charges of basic dyes are able to bind these negative charges, and acidic dyes are repelled.

Since the majority of species in the colon are anaerobes, why might a clinical lab not use anaerobic conditions when identifying stool pathogens?

Because facultative anaerobes and aerobic bacteria are also present in the intestines/colon. In anaerobic conditions, potential stool pathogens that are aerobic bacteria would be unable to grow and would go unidentified in these test

Why do bacterial colonies reach a certain size and then stop growing?

Because of the limited amount of nutrients and resources contained in the broth and/or on the agar plate. Also wastes begin to accumulate.

Which is not a coliform and why? A. Enterobacter aerogenes B. Klebsiella pneumoniae C. Salmonella typhi D. Escherichia coli

C. Pathogen whose infection leads to the development of typhoid, or enteric fever. Coliforms are used to indicate the presence of such a pathogen

Communicable disease

Can be spread from one host to another. This can happen by direct contact between hosts. For example, by droplets (when microbes are carried on liquid drops from a cough or a sneeze), fomites (inanimate objects such as drinking glasses), vectors (insects and other arthropods)

When using the 4X objective is it better to use coarse focus or fine focus?

Coarse focus

MacConkey agar (MAC)

Culture medium selective (only Gram-negative bacteria will grow) and differential (lactose fermenting bacteria form red colonies, lactose non-fermenting bacteria form white colonies). It contains bile salts (to inhibit most Gram-positive bacteria), crystal violet dye (which also inhibits certain Gram-positive bacteria), neutral red dye (which stains microbes fermenting lactose), lactose and peptone

What is a pure culture?

Culture that contains only one type of strain (No contamination by other microbes)

Why is it important that the Petri plates are incubated upside down?

Decreases the chance of microbes landing on the plate and causing contamination. Also, prevents water accumulation due to condensation

Blood Agar Plate (BAP)

Enriched (most bacteria will grow) and differential (for hemolysis) medium used to culture a wide variety of bacteria. It is a TSA plate enriched with mammalian blood (usually sheep), at a concentration of 5-10%. B-hemolytic activity will show lysis and complete digestion of red blood cells surrounding the colony. Alpha-hemolysis will only partially lyse the red blood cells (the hemoglobin and will appear green). Y-hemolysis (or non-hemolytic) is the term referring to a lack of hemolytic activity

Why do you think young cultures should be used when doing a Gram stain?

Ensures that a mixture of gram (+) and gram (-) is shown in a pure culture. If this culture is too old, they may all appear gram (-). This is because as the cells "age" the peptidoglycan is not as tightly cross-linked and gram (+) cells are unable to retain the crystal violet as well

When using the 100X objective is it better to use coarse focus or fine focus?

Fine focus

Trypticase Soy Agar (TSA)

General purpose media produced via enzymatic digestion of soybean meal and casein. TSA is frequently the base media of other agar types; for example, blood agar plates are made by enriching TSA plates with blood

For prep of a smear on a slide, what is the purpose of heat fixation? What problems can arise when the slide is heated in a flame?

Heat fixation adheres the cells to the slide so that they are not washed off later on (e.g during staining). You do not want to overheat the slide because the cells can shrink and the slide will be left with distorted microbes and artifacts.

Why do some genera of bacteria form spores?

In response to unsuitable environmental conditions (ex: high temp, desiccation, extremes in pH, lack of food, etc.) Spores allow these bacteria to exist in extremely low metabolic states until conditions appear that stimulate growth

Pandemic

Increase in disease occurrence within large population over wide regions (usually worldwide)

EMB plates

Inhibit gram (+) growth to a limited degree, and also serve as differential indicators in response to lactose and/or sucrose fermentation

2 members of human microbiota that may live in mouth:

Lactobacillus spp. Bacteroides spp.

Endemic disease

Maintains a relatively steady low-level frequency at a moderately regular interval

What is the major antimicrobial agent in hand sanitizer? How might it work?

Major antimicrobial agent: isopropanol alcohol Works by: break down the microbes cell walls and destroy their ability to regulate their osmolarity, leading to lysis

Explain why methylene blue can't be used in place of nigrosin for negative staining?

Methylene blue is a basic stain (+ charge) that will bind to the negatively charged bacterial cell wall. However, in negative staining the dye should repel the negatively charged bacterial cell wall, leaving the cells unstained against a dark background (use acidic dyes like nigrosin for this)

Are bacterial endospores dead? Explain

No, bacterial endospores are not dead. Endospores are tough, dormant, non-reproducing structures that allow the bacteria to exist in extremely low metabolic states during unfavorable conditions

Are the cells evenly distributed on your slides? Provide an explanation for their distribution.

No, not evenly distributed on the slide. The cells appeared to form clusters which were more concentrated toward one side of the slide. A possible explanation for this is the distribution of nutrients (collected at the bottom of container of water), 3D structure of a biofilm consisting of towers and channels, and H2O flow

Can simple staining be used to identify more than the morphological characteristics of organisms? Explain.

No, simple staining is used to identify cellular shape, size, and arrangement of organisms. The cells are dead, therefore, all metabolic functions have ceased and you cannot study cellular functions.

Can Gram (-), oxidase positive bacteria be identified with the API 20E system test?

No. This system is used to determine the genus and species of enteric bacteria in the family Enterobacteriaceae (gram -, oxidase -). These tests are specific for certain kinds of bacteria and their metabolic activity

Prevalence

Number cases in a population at a particular point in time

Sporadic disease

Occurs occasionally and at irregular intervals

Optochin and bacitracin are used to differentiate catalase negative, Gram positive cocci. How do these chemicals work?

Optochin and bacitracin tests identify whether an organism is susceptible or resistant to optochin and bacitracin. Bacitracin is useful for differentiating B-hemolytic Group A Streptococci from B-hemolytic Non-group A Streptococci. Bacitracin works by disrupting Gram positive bacteria by interfering with cell wall and peptidoglycan synthesis. Optochin is also known as ethylcupreine and is a chemical that inhibits pneumococci but not other alpha reacting streptococci

2 members of human microbiota that may live on hands:

Staphylococcus epidermidis Streptococcus mitis

Epidemic

Sudden increase in frequency above an expected number of classes

Outbreak

Sudden, unexpected occurrence of disease. Usually focal, or in a limited segment of the population

How could you determine if the zone of inhibition is due to death (cidal) or to inhibition (static) of a bacterium?

Swab within the zone of inhibition and streak this swab onto a new non-antibiotic plate. If the antibiotic is bacteriocidal, there will be no growth on the new plate. If the antibiotic is bacteriostatic, there will be growth on the new plate

Which step is the most crucial and the most likely to cause poor results in the Gram stain? Why?

The 10-15 second decolorization step is the most crucial. If the alcohol is left on for >10-15 seconds, both gram (+) and gram (-) cells will be decolorized. If the alcohol is left on for <10-15 seconds, the crystal violet complex may not completely wash out of gram (-)

What bacterial process do you think contributes to making the colon an anaerobic environment?

The aerobic and facultative bacteria in the colon take in any O2 brought to the colon, use it in cellular respiration to produce energy and the by-product CO2. This CO2 accumulates in the colon, making it an anaerobic environment.

What does the term "human microbiota" mean?

The community of bacteria and other microbes that live on or in your body. (Cell paper says that ratio between bacterial and human cells is 1 according to Deighan)

Why is it essential that smears be air-dried?

The liquid will boil when heat fixing the cells, and this boiling will kill the cells. Will denature and lose structure

Incidence rate

The number of new cases in a given time period for the entire at-risk population

How might your streaking technique influence the size, shape and other gross characteristics of bacterial colonies grown on agar plates?

The streaking technique is a way to dilute the microbial sample. Each streak introduces a dilution from the one before and spreads the bacteria until eventually the bacteria are far enough apart to form single colonies. Takes a large, dense sample of patched colonies -> several small, single colonies. If isolated the single colonies, should all look similar

What is the purpose of water in the API tray?

The water creates a humid environment so that the tests do not dry out.

Why are coliforms used as indicator organisms if they are not usually pathogens?

Their presence in H2O indicates the possibility of contamination by other human/animal intestinal pathogens. If these coliforms are detected in H2O, most likely pathogens will be present too. Fecal coliforms are always present in human/animal intestine.

Give an advantage of using blood agar plates for the initial isolation of bacteria?

They are non-selective, therefore, all types of bacteria will be able to grow. These bacteria can be transferred to other agar plates for further isolation and selection.

Why is it necessary to use sterilized growth media?

To kill any existing microbes that entered the media during preparation. If the media is not sterilized, your results will be inaccurate due to contamination by microbes that are not your microbe of interest.

Why are thick or dense smears less likely to provide a good smear preparation for microscopic evaluation?

Will not provide good visualization of individual cells, arrangement and internal structure (gram-staining rxn). Will have the cells packed closely together, right on top of one another, or both.

Index case

first case in an epidemic