Learn: micr141L midterm 1 sample q's

What is an autoclave?

An autoclave uses steam under pressure to achieve sterilization. The lecture gives 15 psi at 121°C for 10-40 minutes. Moist heat denatures proteins; unlike boiling, which is ineffective against spores, an autoclave achieves sterilization.

A chemical compound has a phenol coefficient of 75. Would you consider this a good disinfectant?

Yes, relative to phenol. A phenol coefficient is the reciprocal of the maximum effective dilution of the test disinfectant divided by the reciprocal for phenol; 75 indicates the compound was effective at a dilution 75 times phenol's under the test conditions. It would be considered a good disinfectant by this comparison, but phenol coefficients are only an initial screening and may mislead because they compare two pure strains under specific controlled conditions.

Rank these organisms in the order of toughness (killing them!) mycobacteria, G+, endospores, G-, enveloped viruses

Most resistant to least resistant: endospores of bacteria > mycobacteria > Gram-negative bacteria > Gram-positive bacteria > viruses with lipid envelopes

What advantage does selective and differential media give the clinical microbiologist?

Compared with a general-purpose medium such as TSA, selective and differential media combine selection of growth with differentiation of growth types. The lecture labels MacConkey agar as selective and differential, while TSA is general-purpose and blood agar is differential only; this helps focus interpretation and further culture work-up.

What is the purpose of controls in gram staining? When do you use these?

Because Gram staining is differential, controls provide known results for comparison with the unknown and help validate the stain. Use the controls with the Gram-stain run: Staphylococcus aureus should be G+ cocci in clusters, and Escherichia coli should be G- rods with no arrangement. Appropriate controls are one of the criteria for a valid Gram stain.

What are the steps of gram stain? What happens at each step?

Prepare a thin smear and fix it; heat fixation is routine for prokaryotes. In the Gram-stain sequence represented by the lecture's principles, crystal violet is retained by G+ cells because it binds teichoic acid; iodine replaces chloride in the stain molecule with iodine and links it to the cell wall; alcohol-acetone damages the thin lipid wall of G- cells and washes out crystal violet; safranin then stains nonstained elements pink.

What is the difference between heat fixing and alcohol fixing a smear?

The lecture specifically contrasts heat fixation with chemical fixation rather than giving a separate alcohol protocol. Heat fixation is routine for prokaryotes and preserves overall morphology but not internal structures. Chemical fixation, the category used for the alcohol method named in the question, is described as protecting fine cellular substructure and morphology in larger, more delicate organisms. Fixation preserves and positions structures, and organisms are usually killed and firmly attached to the slide.

What is TSA used for?

TSA, or tryptic soy agar, is a general-purpose growth medium. The lecture's recipe is casein peptone 15.0 g, soybean peptone 5.0 g, sodium chloride 5.0 g, and agar 15.0 g per liter.

When is direct gram staining of a patient's sample useful?

Direct Gram staining gives rapid direct-examination information: it confirms that the submitted material is representative; identifies cellular components and inflammatory debris to estimate the probability of infection; may identify specific infectious agents; guides early antibiotic treatment; and can develop epidemiologic data.

The physician has indicated that (s)he needs a gram stain of the culture as soon as plates have grown. How would this help the patient?

A Gram stain of a newly grown culture provides a preliminary report: morphology and Gram reaction help interpret the specimen, select potential pathogens, and decide on further testing. A presumptive identification based on morphology can support empirical antimicrobial treatment while the culture work-up continues.

Swabbed specimens are sent in culturettes. Generally what is found in the culturette tube? How does this help in specimen transport?

A culturette generally contains a transport or holding medium around the swab. Transport or holding media maintain the viability of microorganisms present without supporting growth; examples in the lecture include Stuart's and Amies media. This preserves the specimen during transport without allowing organisms to multiply in the tube.

Why do you not want an old specimen for culturing?

An old specimen is undesirable because viability can be lost during transport; the lecture says there is always some loss of viability and transport time should be minimized. Without appropriate preservation or with prolonged transport, the specimen may be rejected or may no longer support an accurate culture result.

List five factors that have a significant role in the selection and implementation of an appropriate method of disinfection.

Five important factors are the number of microbes, type or characteristics of the microbes, concentration of the antimicrobial agent, exposure time, and temperature. The lecture also lists pH, organic matter, and biofilm formation; selection additionally considers required sterilization, heat/pressure/radiation/chemical tolerance, suitability, penetration, cost and labor efficiency, and safety.

How are antiseptics and disinfectants different?

Disinfection removes or reduces pathogens on an object or surface and does not necessarily remove or kill all microbes. Antisepsis is removal of pathogens from living tissue; an antiseptic is a mild disinfectant suitable for use on skin surfaces.

What is immersion oil used for?

Immersion oil is used between the microscope slide and the 100x oil-immersion objective. It reduces the refraction or bending of light as it passes through glass, allowing maximum use of the objective's numerical aperture; with a 10x ocular and 100x oil objective, total magnification is 1000x.

What type of information do you expect to be included with a specimen? Why?

A specimen should be accompanied by patient identification; clinical data when indicated; requesting physician or authorized person's contact information; tests requested; date and time of collection; and specimen source when appropriate. This information supports positive identification, correct testing and interpretation, communication with the requester, and assessment of timing and source.

What is wrong with using an old culture for gram staining?

An old culture violates a criterion for a valid Gram stain: the lecture requires a young culture, 18-24 hours. The lecture does not specify the mechanism of the old-culture error, but it indicates that the result may not be considered valid unless the culture is young, the smear is thin, reagents are proper, and controls are appropriate.

A patient has had an infection for 7 days. Why is it that doing a direct gram stain for this patient does not violate the "young culture" rule?

The 18-24-hour young-culture criterion refers to a laboratory-grown culture. A direct Gram stain is made from the patient's specimen itself, not from a seven-day-old laboratory culture; its purpose is rapid direct examination and early information about infection. Therefore, the patient's seven-day illness does not violate the culture-age criterion.

Why do we use methylene blue as secondary dye in acid fast stain, but we do not use that as secondary dye in Gram stain?!

pink (safranin) and purple contrast better than blue (methylene blue) and purple do. they chemically would work the same, but it is just easier to differentiate pink and purple as blue is too similar to purple.

Errors in the laboratory are usually the result of...

Errors often result from specimen-management pitfalls: saying yes to everything and accepting every specimen, being afraid to say no to physicians, and having no boundaries for technical issues. These can lead to delays, unnecessary redraws or retests, decreased customer satisfaction, increased costs, incorrect diagnosis or treatment, injury, or death.

What are the different biosafety levels?

BSL-1: agents not known to consistently cause disease in healthy adults. BSL-2: agents associated with human disease; hazards include percutaneous injury, ingestion, and mucous-membrane exposure. BSL-3: indigenous or exotic agents with potential for aerosol transmission and serious or lethal disease. BSL-4: dangerous or exotic agents with high risk of life-threatening disease, aerosol-transmitted laboratory infections, or unknown transmission risk. The BSL indicates infection potential and engineering controls used to protect workers.

Cary-Blair medium contains salt. Why? It contains sodium thioglycollate, why?

NaCl ensures osmotic and structural stability.

Sodium thioglycollate ensures a reducing environment that protects against oxidative damage.

What is the function of Resazurin in an anaerobe transport vial? Cysteine?

Cysteine is the chemical that creates the anaerobic environment, and resazurin is a redox indicator that indicates that there is indeed an anaerobic environment. This ensures the viability of anaerobic microorganisms during transport

How should a collected specimen be transported to lab (discuss time, and transport condition)?

Use appropriate safety and packaging, preservation, temperature, acceptable transport time, and transport mode; personnel must be trained, specimens are handled as infectious, and IATA regulations are followed when applicable. Minimize transport time or use the recommended transport medium because some viability is always lost. Lecture examples: without medium, process all specimens within 1 hour; stool culture 2 hours or Cary-Blair 48 hours; GC swabs immediately into Amies with charcoal and no more than 24 hours in that medium at ambient temperature; respiratory viral specimens up to 48 hours in viral transport medium; C. difficile toxin specimens 2 hours at ambient, 72 hours at 4°C, or 1 week frozen; urine for CMV 24 hours stored at 4°C.

Sometimes you'll have to send specimen to reference laboratories. What are these?

Reference laboratories areor each referral, record the tests or specimens, date of referral, and person referring; then track turnaround time, delivery of results from the referral lab to the requester, and any problems.

List some ways you might be exposed to pathogens in lab.

Exposure routes listed for BSL-2 hazards are percutaneous injury, ingestion, and mucous-membrane exposure. BSL-3 and BSL-4 concerns also include aerosol transmission and laboratory infections. Thus, exposure can occur through a puncture or other percutaneous injury, swallowing, mucous-membrane contact, or infectious aerosols.

Be familiar with type of collection devices used for various body sites.

Use the device appropriate to the specimen type or site: swab specimens go in a swab transport system such as Amies or Amies with charcoal; GC swabs specifically go immediately into Amies with charcoal or another approved GC transport medium. Stool cultures may use Cary-Blair; fluid specimens are collected in a collection cup; blood cultures use aerobic and anaerobic bottles; and anaerobic specimens use an anaerobic transport system or medium. The lecture shows these devices but does not provide a complete body-site table.

What is the goal of hand washing (be specific)?

Get rid of most bacteria?

Why would you expect 2 swabs if a stat gram stain is requested along with ID?

Two swabs are needed because one is used to prepare the direct Gram smear and the other is used to inoculate culture media for identification and work-up. The lecture says never use the swab used to inoculate media for the smear, always collect two swabs, roll the smear rather than rub it, and preserve morphology and organism relationships.

Why is it important to collect a specimen prior to antibiotic treatment?

The lectures do not explicitly state the antibiotic-specific reason. They establish that the specimen is essential to accurate diagnosis and directly influences treatment, and that direct examination can guide early antibiotic treatment. Thus, within the lecture's logic, collect before treatment so the diagnostic specimen is available before treatment can interfere with the clinical work-up; the exact mechanism is not given.

What are the risks of being too eager to please the customer (saying yes to everything)?

Being too eager to please can mean accepting every specimen, being afraid to say no to physicians, or having no technical boundaries. The lecture says this can cause delays, unnecessary redraws or retests, lower customer satisfaction, higher costs, incorrect diagnosis or treatment, injury, and death. Good laboratory practice keeps patients first and requires authority to support policy.

Why do you need to wipe down your bench-top before and after work?

Before: Get rid of majority of pathogens to ensure work does not get contaminated. After: Ensure that nothing you worked with contaminates the bench-top.

Know the Gram reaction and morphology of: E. coli, Proteus spp., S. aureus, Bacillus spp., Micrococcus spp.

Escherichia coli: Gram-negative rods; Staphylococcus aureus: Gram-positive cocci in clusters. Proteus spp.: Gram-negative rod-.: shaped in short chains or single (motile on agar). Bacillus spp.: Gram-positive rod-shaped in single, pairs, of chains. Micrococcus spp.: Gram-positive cocci in tetrads.

A specimen has a life before, and after you. What are the important points in collection? Transport? What happens after we are done with the specimen (think storage and eventual discard)?

Collection: positively identify the patient; use the required specimen, container, volume, and timing; prepare the patient; use preservation or transport medium if needed; label properly; and follow special handling. Transport: train personnel in safety and packaging; package and preserve correctly; use appropriate temperature, acceptable time, and transport mode; and follow IATA. Handling: treat all specimens as infectious; log or accession them and confirm receipt, date, and time; track aliquots; and have procedures for stat or delayed tests, leaking containers, contaminated forms, and preservation. Referral: record test or specimen, date, and referrer; track turnaround, result delivery, and problems. Storage: establish policy, retention time, location and conditions, indexing, organized tracking, and freeze-thaw monitoring. Disposal: follow medical-waste and disinfection policies, local regulations, rejected-specimen procedures, oversight, and scheduled review of stored specimens.

Why do we not want a transport medium in a urine collection cup? What does this lack of a transport medium mean for urine specimen?

Transport medium for urine increases risk for contamination, and can interfere with the lab testing. Since it is without transport medium, it should generally be processed within 1 hour; the table i the lecture slides separately lists urine for CMV as 24 hours stored at 4°C and says transport medium is not recommended.

If given a recipe for a medium (the ones covered so far) be able to assign a function to each.

Lecture-supported medium functions: TSA is a general-purpose growth medium; blood agar is differential only; MacConkey agar is selective and differential; Cary-Blair, Amies, and anaerobic transport media are transport or holding media that maintain viability without supporting growth; charcoal in transport media absorbs fatty acids so fastidious organisms such as Neisseria gonorrhoeae or Bordetella pertussis can grow; the fecal transport vial is non-nutrient, buffered to 7.7, and uses phenol red as an indicator; and Löwenstein-Jensen medium is shown with malachite green, glycerol, asparagine, potato starch, coagulated eggs, and mineral salts, but individual ingredient functions are not given. The lecture explicitly says blood-agar casein enzymic hydrolysate and yeast extract provide nitrogen, carbon, amino acids, and vitamins; for other individual ingredients, it supplies recipes but not functions.

What are flagella? Can we see these in a gram stain?

Flagella are bacterial structures shown with a filament, junction zone, hook, basal body and stators, and export apparatus. The lecture shows four arrangements: monotrichous (one), lophotrichous (multiple from one end), amphitrichous (one each end), and peritrichous (mr. worldwide).

What are endospores? How do we visualize these? Who makes them?

Bacterial endospores are structure made by some gram+ bacterial species that are highly resistant. boiling is ineffective against spores, and sterilization includes spores. We can do an endospore stain using malachite green which can stain them while safranin stains only non-endospore cells. We worked with Bacillus which could form endospores.

How are TSA and blood agar similar? Different? Given the recipe, can you assign a function to ingredients of a given medium?

TSA and blood agar are both solid growth-media examples containing nutrient ingredients, sodium chloride, and agar. TSA is labeled a general-purpose growth medium and contains casein peptone 15.0 g, soybean peptone 5.0 g, sodium chloride 5.0 g, and agar 15.0 g per liter. Blood agar is labeled differential only and contains casein enzymic hydrolysate 14.0 g, peptic digest of animal tissue 4.5 g, yeast extract 4.5 g, sodium chloride 5.0 g, agar 12.5 g, and sheep blood 5.0 g per liter; the lecture says casein enzymic hydrolysate and yeast extract provide nitrogen, carbon, amino acids, and vitamins. The key listed difference is sheep blood and the differential-only designation.

How much agar is in a solid growth medium? How about a broth?

The solid growth-media examples in the lecture contain about 1.25-1.5% agar: TSA 15.0 g/L (1.5%), blood agar 12.5 g/L (1.25%), and MacConkey 13.5 g/L (1.35%). The uploaded lectures do not give a broth recipe; a broth would have no agar, but that exact comparison is not directly shown. Cary-Blair (5.0 g/L), Amies (4.0 g/L), and fecal transport medium (0.15% w/v) contain less agar than these solid growth-media examples.

Mycobacteria....what do they cause? How do we stain these?

Mycobacterium tuberculosis causes tuberculosis, and M. leprae causes leprosy or Hansen disease. The lecture also lists M. scrofulaceum with cervical lymphadenitis in children, M. avium intracellulare with disseminated disease in immunocompromised people, M. kansasii with lung disease, and M. marinum with cutaneous disease secondary to water exposure. The lecture states that all Mycobacterium shown are acid-fast. For the Kinyoun acid-fast stain, cover with carbolfuchsin for 5 minutes, wash, decolorize with acid alcohol until the color is removed, wash, flood with methylene blue for 1 minute, wash, and air-dry; carbolfuchsin binds to mycolic acid.