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Questions and Answers

What effect does alpha hemolysis have on red blood cells?

Complete destruction of red blood cells

Only affects platelets

Incomplete hemolysis producing a greenish discoloration (correct)

No effect on red blood cells

What temperature must chocolate agar be when the blood is added for preparation?

37°C

80°C

72°C (correct)

50°C

At what temperature does agar set after being melted?

80°C

37°C

98°C

42°C (correct)

What is the simplest way to test the pH of a culture medium?

Using narrow range pH papers or a pH meter Signup and view all the answers

What is the main purpose of incubating 5% of a media batch at 35–37 °C?

To test for sterility by checking for contamination Signup and view all the answers

What percentage of agar is needed to create semisolid media?

0.6% Signup and view all the answers

What happens to agar when placed in water before it is heated?

It remains as solid lumps Signup and view all the answers

What characterizes gamma hemolysis?

No change or effect on red blood cells Signup and view all the answers

What is the primary purpose of fixation in microbiological smears?

To preserve microorganisms and prevent washing away during staining Signup and view all the answers

Which method of fixation is considered less damaging to microorganisms?

Alcohol fixation Signup and view all the answers

For how long should heat-fixed smears be held over the flame during fixation?

Which of the following statements about heat fixation is accurate?

It can leave some organisms, like M.tuberculosis, alive. Signup and view all the answers

What technique is recommended for making smears from purulent specimens?

Use a sterile wire loop to create a thin preparation. Signup and view all the answers

How long should alcohol be left on a smear for proper fixation?

For a minimum of 2 minutes or until evaporated Signup and view all the answers

Which of the following statements is true regarding non-purulent fluid specimens?

A smear is made from a drop of well-mixed sediment. Signup and view all the answers

What is the recommended method for transferring caseous material from sputum to a slide?

Roll a clean stick over the material and then on the slide. Signup and view all the answers

What is one of the main purposes of buffered charcoal–yeast extract agar (BCYE)?

To provide nutrients for the growth of Legionella pneumophila Signup and view all the answers

What is the selective property of MacConkey media?

Inhibits gram positive bacteria growth Signup and view all the answers

What color change indicates lactose fermentation on MacConkey media?

From colorless to pink Signup and view all the answers

Which of the following statements about Mannitol Salt Agar (MSA) is true?

It is selective due to its high NaCl concentration. Signup and view all the answers

What distinguishes Eosin Methylene Blue (EMB) agar's differential property?

It differentiates between bacteria based on the color of colonies. Signup and view all the answers

Which type of hemolysis is indicated by a clear area around a bacterial colony on blood agar?

Beta hemolysis Signup and view all the answers

What function does blood agar primarily serve in a clinical microbiology lab?

It is an enriched medium for fastidious organisms. Signup and view all the answers

What type of bacteria does MSA effectively identify?

Pathogenic Staphylococci Signup and view all the answers

What is the primary purpose of the Gram stain procedure?

To classify bacteria into two groups based on their cell wall structure Signup and view all the answers

Which component of the Gram stain serves as the primary stain?

Crystal violet Signup and view all the answers

What color do gram-positive bacteria appear after the Gram staining process?

Dark purple/Blue Signup and view all the answers

What is the effect of the decolorizing agent in the Gram staining process?

It washes out the purple dye from gram-negative bacteria Signup and view all the answers

Why is rolling the swab important when collecting samples for Gram staining?

To avoid damaging the pus cells Signup and view all the answers

What color do gram-negative bacteria appear after the Gram staining procedure?

Red to pink Signup and view all the answers

In which scenario should a report indicate 'No organisms seen'?

If no cells or organisms are detected in a clinical specimen Signup and view all the answers

What role does safranin play in the Gram staining process?

It counterstains the decolorized gram-negative bacteria Signup and view all the answers

What is indicated by a positive Citrate test?

An organism can utilize citrate as a carbon source Signup and view all the answers

Which result indicates that the organism is urease positive?

Blue or purple color change Signup and view all the answers

What does a positive indole test indicate about an organism?

The organism produces indole from tryptophan Signup and view all the answers

What does a positive MR test suggest about an organism's metabolic capabilities?

It favored mixed acid fermentation Signup and view all the answers

Which statement is true regarding the VP test?

It indicates the presence of acetoin Signup and view all the answers

What would a positive result in the H2S test indicate?

The organism produces hydrogen sulfide Signup and view all the answers

What does a blue or dark purple color in an oxidase test suggest?

The organism produces cytochrome oxidase Signup and view all the answers

In the TSI test, what does K/A mean?

No carbohydrate fermentation Signup and view all the answers

Which method provides an indirect measurement of cell density, only related to live bacteria?

Plate count method Signup and view all the answers

What is the acceptable range for colony counts on a plate to ensure statistical reliability?

25 to 250 colonies Signup and view all the answers

What does CFU stand for in microbiology?

Colony-forming unit Signup and view all the answers

Which substance is NOT part of the materials used in the Kirby-Bauer method?

Dilution buffer Signup and view all the answers

What is the function of the McFarland Standards in the laboratory?

To standardize turbidity for bacterial counting Signup and view all the answers

What happens if plates have fewer than 25 colonies?

They are often disregarded for accuracy Signup and view all the answers

What environment is the Mueller-Hinton agar considered the best for?

Routine susceptibility testing of non-fastidious bacteria Signup and view all the answers

What is the crucial first step when using antibiotic disks in susceptibility testing?

Allowing the disks to warm up Signup and view all the answers

Study Notes

Laboratory Safety Procedures

Report all broken glassware to the instructor
Follow instructions for cleanup
Practice aseptic transfer methods
Wash hands before and after lab work, and when contamination is suspected
Do not eat or drink in the lab
Disinfect lab benches before and after each lab session
Properly dispose of lab waste
Do not apply cosmetics, handle contact lenses, or place objects in your mouth
Read and sign the lab safety agreement
Properly return materials to their designated locations
Use appropriate labels on specimens and instruments
Train faculty and staff on Material Safety Data Sheets (MSDS)

Protective Procedures

Tie back long hair
Wear appropriate personal protective equipment (eye protection, coats, closed shoes)
Use appropriate pipetting devices
Do not use mouth pipetting

Emergency Procedures

Know the location and proper use of emergency equipment (eye wash stations, fire extinguishers, chemical safety showers, and emergency numbers)
Report all injuries immediately to the instructor and follow instructions
Follow proper steps in the event of an emergency

Safety Data Sheets (SDS)

SDSs (Material Safety Data Sheets) are forms providing information on substance properties
Includes physical data (melting point, boiling point)
Information on toxicity, reactivity, health effects, storage, and disposal

Personal Protective Equipment (PPE)

PPE provides an immediate barrier between personnel and hazardous agents
Items include lab coats, gowns, gloves, goggles, face masks, and foot protection

Laboratory Safety Equipment

Safety showers
Eye wash stations
Fire blankets
Fire extinguishers
Sharp containers for disposal of sharps (tips, slides, broken glass, glass tubes, bottles, scalpels, and needles)

Biological Safety Cabinets (BSC)

BSCs enclose workspaces to protect workers from aerosol exposure to infectious agents
Air is sterilized by heat, ultraviolet light, or HEPA filtration
Removes particles larger than 0.3 mm in diameter
Type II A BSCs are commonly used in clinical labs for processing specimens and isolating agents
Type I hoods are rarely used
Type III hoods are not commonly used in diagnostic labs

Biosafety Levels (BSL)

BSL-1: Basic protection for agents not known to cause disease in healthy humans
BSL-2: Protection for moderate-risk agents known to cause human disease
BSL-3: Protection for agents with a high potential for aerosol transmission
BSL-4: Protection for exotic agents posing a high individual risk of life-threatening disease, no known treatment exists

Microbiology Laboratory Waste Disposal

All materials contaminated with potentially infectious agents must be decontaminated before disposal.
This includes patient specimens, cultures, sharp instruments (e.g. glass slides, tubes, scalpels, syringes with needles), unused reagents.
Decontamination may be done via autoclaving, incinerating, or other methods.
Some municipalities allow specific waste types like blood, serum, urine, feces, and body fluids to be poured into sanitary sewers, while other infectious waste is autoclaved or incinerated.
Infectious waste should be double bagged (placed in leak-proof, plastic bags) for added sturdiness

Instruments and Tools Used In Microbiology Labs

Bunsen burner
Swabs
Hot plate
Incubator
Petri dish
Autoclave
Wire loop
Needle
Anaerobic jar
Gas generator envelopes
Anaerobic indicator strip

Observing Microorganisms Through a Microscope

Microscopy magnifies small objects to be easily viewed
Units of measurement used include cm, mm, μm, nm, and Å
Common types of microscopy include bright-field, fluorescence, dark-field, and electron

Oil Immersion Techniques

Using mineral oil between the lens and slide prevents light loss
Improves resolution

Darkfield Microscopy

Used to view live microorganisms that are invisible in ordinary light microscopes
Cannot be stained by standard methods
The specimen appears bright against a dark background

Fluorescence Microscopy

Used to detect specific antigens rapidly
Uses UV light
Fluorescent materials absorb UV light and emit visible light

Electron Microscopy

Used for objects smaller than 0.2 μm
Uses electrons instead of light for higher resolution
Images are always black and white.

Direct and Indirect Smears

Direct smears: a preparation of the primary clinical sample
Used to identify cells (WBCs, epithelial cells) and organisms
Indirect smears: preparation from organisms grown in culture
Provides identification after purification and growth using artificial media.
Care taken in preparing smear from solid and liquid media

Preparation and Staining

Wet mount: observing live microbes, their shape, and arrangement
Hanging drop: differentiating between motile and nonmotile microorganisms

General Concepts for Specimen Collection and Handling

Properly collect specimens before administering antimicrobial agents.
Select appropriate anatomic sites and sufficient specimen quantity.
Adhere to collection and transportation instructions.
Label specimens with clear patient information.
Use sterile or non-sterile collection devices as needed.
Transport specimens to the lab within 2 hours of collection.
Use leak-proof containers.
Mark containers with biohazard labels
Preserve specimen viability by using appropriate preservatives (e.g., boric acid, PVA, formalin).
Store preparations at appropriate temperatures, protected from light.

Media Preparation

Media are mixtures of nutrients to support microbial growth on a solid (agar) or liquid (broth) form.
Types of bacterial media include nutritive, selective, differential, and enrichment media.
Nutritive: Support growth of most organisms without selective advantages
Selective: Inhibits certain types/group of organisms (e.g., gram-positive microbes), favoring the growth of others.
Differential: Distinguish between various microbes that grow on the same medium.
Enrichment: Enhance the growth of specific microbes/pathogens

Media Classifications Summary

Nutritive: Supports most non-fastidious organisms
Selective: Inhibits growth of some microbes, favoring the growth of others
Differential: Distinguishes between different microbes
Enrichment: Enhances the growth of specific microbes

Cultivation and Isolation of Bacteria (Streaking)

Streak plates are used to obtain isolated colonies
3-4 quadrant methods
Sterilize the inoculating loop in a Bunsen burner flame, allow it to cool.
Transfer isolated colony from the agar plate to the first quadrant
Continue streaking into other portions using separate, parallel streaks or strokes
Put plates in an incubator for pure growth

Cultivation and Isolation of Bacteria (Test Tubes)

Ways to culture bacteria from solid media
Slant media : Plunge wire into the center of the medium.
Stab media : Plunge wire into and streak surface of medium using wire in a zigzag pattern.

Staining Methods

Heat-fixed smear with crystal violet (primary stain)
Cover smear with iodine (mordant)
Wash with alcohol (decolorization)
Counterstain with safranin

Gram Stain

Classifies bacteria into gram-positive (stains purple) and gram-negative (stains pink/red).
Based on cell wall differences

Ziehl-Neelsen

Identifies acid-fast bacteria in clinical samples (e.g., Mycobacterium tuberculosis, M. leprae)
Bacteria retain the primary stain (carbolfuchsin) even after decolorization, then visualized in Red color
Non-acid-fast bacteria appear blue after counterstaining.

Bacterial Identification Using Phenotypic Criteria

Microscopic (shape, arrangement, Gram stain)
Macroscopic (colony morphology, odor, pigmentation)
Environmental requirements (temperature, atmosphere)
Resistance/susceptibility to antimicrobial agents
Biochemical reactions (enzymatic tests)

Colony Count

Used to estimate the number of viable bacterial cells in a sample.
Performed by diluting the sample, spreading the diluted sample onto plates for growth.
Counting colonies that grow helps estimate original level of bacterial cells.

Antimicrobial Agents (Kirby-Bauer Method)

Used to determine the susceptibility of bacteria to various antimicrobial agents.
A standardized method using Mueller-Hinton agar plates
Antimicrobial disks are placed on the agar plate containing the test bacteria.
Measure the zone of inhibition around the disks to determine drug sensitivity or resistance.

Biochemical Tests

Used to identify bacteria based on their metabolic characteristics
Multiple tests are routinely used as part of the process

Diagnostic of the Digestive System Infections

Stool samples
Rectal swabs
Used for determining pathogen causing diarrhea or other issues
A wide variety of bacteria can cause infections.
Specimen collection needs a sterile, wide-mouth container, and delivered within 1 hour
Some pathogens, such as Shigella, are susceptible to lower temperatures, so refrigerating specimens is not recommended

Diagnostic of the Respiratory Tract Infections

Throat swabs
Used for infections of the upper and lower respiratory tracts
Specimen collection, and process immediately, or refrigerate if necessary, send to lab within 2-4 hrs

Specimen Collection and Transport

Collected in sterile, wide-mouthed containers for stool and vaginal cultures.
Should be sent to the lab promptly to prevent microbial changes

Direct Microscopic Examination (Gram Stain & Wet Mount)

Used to observe organisms directly from samples (e.g. vaginal, urethral swabs)
Gram stains are used to identify gram positive vs gram negative microorganisms
Smears can be made from fluid specimens or swabs

Urinary Tract Infection (UTI)

Urine sample collection (Clean catch, midstream)
Proper transport of urine specimen within 2 hrs. (optional refrigeration at 2-8°C for longer transport)
Routine bacterial culture, performed on blood agar, MacConkey, CLED agar to help distinguish between contamination and infection.
Colony counts determine level of infection

Blood Culture

Use strict aseptic techniques for collections and transport.
Multiple blood samples are collected over 24 hrs or more (in case of suspected bloodstream infection)
Collect and process without delay to avoid contamination

Spinal Fluid Culture

Used for diagnosing meningitis
Collect and process the specimen without delay

Eye Culture

Specimens collected using a Dacron swab
Culture using blood agar, chocolate, and MacConkey for identification and antimicrobial sensitivity
Look for bacterial infections or presence of pus cells in specimen

Other Methods

Physical mechanisms of sterilization (Heat, Filtration, Irradiation, Cooling, Metallic Salts, Ultrasonic/Sonic Vibration)
Disinfection methods (Phenol, Halogens, Alcohols, Heavy Metals, Aldehydes)
Fungi Culture and Microscopy (Yeasts & Molds)

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