Untitled Quiz

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)