Copyright © 2010 Delmar, Cengage Learning. ALL RIGHTS RESERVED. Chapter 43 Basic Microbiology.

Copyright © 2010 Delmar, Cengage Learning. ALL RIGHTS RESERVED.

Chapter 43

Basic Microbiology


The Medical Assistant’s Role in the Microbiology Laboratory

• Preparing cultures

• Allow bacteria to grow at least 12 hours before examining culture

• Sensitivity identifies which antibiotics will kill microorganism causing infection



• Use exact technique to avoid laboratory error

• Use sterile equipment

• Send culture to laboratory in reasonable amount of time



• Identification of organisms done successfully within 24–72 hours


Microbiology

• Bacteria are found naturally in the body– Normal flora

– Always present and help with immune system

– Pathogens cause disease


Microbiology

• Classification– Taxonomy deals with classification of living organisms

– Carolus von Linnaeus devised current classification system

– No universal agreement on one system


Microbiology

• Classification– Kingdoms

• Plants

• Animals

• Protists– Prokaryotes (lower protists)

– Eukaryotes (higher protists)


Microbiology

• Nomenclature– System for naming bacteria

– Genus• First name; capitalized

– Species• Second name; not capitalized


Microbiology

• Nomenclature– Bacteriologists and microbiologists

– Parasitology

– Virology

– Mycology

– Reference laboratory

– Report certain types of bacteria and yeasts to Department of Public Health


Microbiology

• Cell structure– Basic bacterial cell >>

– DNA (deoxyribonucleic acid)


Equipment

• Autoclave– Used to sterilize equipment

– Not used with presterilized and disposable equipment


Equipment

• Microscope– Used to view

organisms that cannot be seen with naked eye

– Prepared slides used


Equipment

• Safety hood– Aerosols can be released into air when culturing and

are potentially dangerous if inhaled

– Use of hood is mandatory when performing culture on specimen with potential aerosol

– Used to minimize odors


Equipment

• Incubator– Has constant temperature of 35–37°C

– Grows aerobic or anaerobic organisms

– When culturing, set up some cultures in oxygenated environment as well as oxygen-reduced environment


Equipment

• Anaerobic equipment– Absence of oxygen to grow anaerobic bacteria

– Use of candle jar

– Gas pack jar

– Specimens sent to reference laboratories

– Gram stain used to observe gross morphological features of bacteria


Equipment

• Inoculating equipment– Inoculating loop

– Inoculating needle

– Stab culture used for certain biochemical tests used for identification


Equipment

• Incinerator– Quickest method of sterilization

– Electrical incinerator or Bunsen burner

• Media– Host of substances

– Used to foster growth of bacteria


Equipment

• Refrigerator– Used to store materials

– Temperature of 2–8°C

– Never store food or drink or medication with specimens


Safety When HandlingMicrobiology Specimens

• Personal protector when handling microbiology specimens– Wear PPE at all times

– Remove when leaving for the day

– Buttoned laboratory coat or apron, safety goggles, and gloves



• Personal protector when handling microbiology specimens– Use of hood or shield

– Never eat, drink, smoke, or put objects into mouth

– Do not touch contact lenses or apply makeup

– Wash hands frequently



• Work area– Use strong germicide before and after daily use or

immediately after spills

– Dust-free and clean at all times

– Uncluttered

– Avoid body burns or files



• Specimen handling– Check for leaks and contamination on containers

– Wear gloves

– Use appropriate container

– Handle all specimens as if contaminated



• Disposal of waste and spills– Biohazard symbol

– Separation of biohazardous wastes

– Disinfect spills with 10 percent bleach solution


Quality Control

• All equipment with temperature controls should be monitored daily

• Microscopes should be cleaned and kept dust-free

• Media of all types should not be used past shelf life– Should be stored at proper temperatures– Checked for growth with known organisms for quality

control


Quality Control

• Procedure manual with all standard operating procedures written down should be updated periodically

• Many microbiology laboratories subscribe to associations that periodically send unknown samples to be set up and identified


Collection Procedures

• Check to see if culture was:– Collected properly

– Delivered within a reasonable period of time

– Collected in sufficient quantity



• Common microbiology specimen sites

• Place in appropriate container

• Bring to laboratory

• Rejecting specimens



• Factors determining successful isolation of causative pathogens– Proper collection from infection site

– Collection of specimen during infection period

– Sufficient amount of specimen

– Appropriate specimen container

– Appropriate transport medium



• Factors determining successful isolation of causative pathogens– Specimen labeled properly

– Specimen brought to the laboratory in a minimal amount of time

– Specimen collected before administration of antibiotics

– Specimen inoculated onto proper media and placed in correct atmosphere to ensure growth


Specific Collection Requirements

• Urine– Collecting a clean-catch specimen

– Use of catheterization

• Nose– Nasal-pharyngeal swab collects specimen

– Place swab in sterile tube for transport to laboratory



• Throat– Use sterile tongue depressor to hold patient’s tongue down

– Avoid swabbing sides of mouth and tongue



• Wound– Use of sterile needle or swab to aspirate pus-filled fluid

from wound

– Use of anaerobic transport medium



• Sputum– Patient coughs deeply and expectorates into sterile

container

– Should be morning specimen

– Use of special container



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• Stool– Ova and parasites

– Bacterial cultures

– Non-sterile containers

– Contamination of urine



• Cerebrospinal fluid (CSF)– Lumbar puncture

– Fluid dispersed in several departments of clinical laboratory

– Use of incubator

– Refrigeration can kill meningitis-causing bacteria



• Blood– Development of septicemia

– Collection of cultures

– Variety of collection devices available


Bacterial ShapesCocci Bacilli

Spirilla


Microscopic Examination of Bacteria

• Dyes– Derived from coal tar

– Acidic dyes carry a negative ion

– Basic dyes carry a positive ion

– Methylene blue binds to DNA and RNA of cell



• Simple stain– Uses single stain on fixed slide for given period of time

– Shows structure and arrangement of bacterial cell

– Takes no more than 3 minutes to stain

– Gives little information other than size and morphological arrangement



• Differential stain– A common differential stain is the gram stain

– Use of decolorizer and counterstain

– Developed in 1884 by Dr. Hans Christian Gram

– Differentiates bacteria by gram stain ability of being negative or positive

– Use of gentian or crystal violet reagents



• Differential stain– Identifies gram-positive and gram-negative bacteria

• Staphylococcus

• Streptococcus

• E. coli

• Proteus

– Morphological arrangement, shape, and gram-stain characteristic help identify bacteria



• Acid-fast stain– Specific stain

– Allows microscopic examination of acid-fast mycobacteria

– Use of heat or powerful dye

– Ziehl-Neelsen stain

– Kinyoun stain



• Special techniques– Used when flagella, spore, capsule, or nuclei of cells

are present

• Tests without staining– Wet slide preparation

– Hanging drop



• Potassium hydroxide (KOH) preparation– Used for study of fungi and spores

– Fragments of human hair, skin, or nails placed on slide with drop of 10 percent KOH and coverslip

– KOH clears debris



• Potassium hydroxide (KOH) preparation– Set slide at room temperature for one-half hour before

examination for debris settlement

– Use of phase or dark-field microscope

– Dispose of properly (live organisms)

– Direct microscopic examination of culture and infectious bacteria


Culture Media

• Inoculate material on proper medium for growth

• Reliability of results

• Fastidious bacteria need specialized medium to grow

• Aerobic bacteria grow only in oxygen


Culture Media

• Common bacteria and growth requirements

• Transport media

• Can be solid, liquid, or semisolid substance


Culture Media

• Contains nutrients to support growth of bacteria– Vitamins

– Sugar

– Salt

– Minerals

– Amino acids

– Addition of special products


Culture Media

• Agar– Solid media

– Poured in petri dish or tubes

• Broth tubes store semisolid media


Culture Media

• Media classification– Basic

– Differential

– Selective

– Enriched

• Common specimens, suspected pathogens, media recommendations


Microbiology Culture

• Inoculating the media– Roll swab onto upper quadrant of agar plate

– Use loop to inoculate sputum or liquid

– Spread flamed loop or needle back and forth in sweeping motion to dilute bacteria



• Inoculating the media– After inoculating agar plate, turn upside down and

place in proper environment for growth

– Broth tube inoculation

– Deep inoculation/slant



• Other types of streaking– Lawn streak used to place organism over entire area of

agar plate for sensitivity testing

– Colony count used to plate urine cultures

– Laboratories have slightly different ways of performing basic streaks



• Primary culture– Read after media incubated 24–48 hours

– Observe for gross colony characteristics• Size

• Shape

• Color

• Elevation



• Primary culture– Observe for gross colony characteristics

• Density

• Consistency

• Hemolytic versus nonhemolytic

• Odor

• Pigment production

– Use of bright, direct light



• Subculture– More than one pathogen grows in culture

– Separation of pathogenic bacteria from normal flora• Use of inoculation loop or needle

• Pick suspicious pathogen and streak onto media

• Allow to grow for 24 hours


Identification Systems

• Streptococcus screening– Rapid identification important

– Many rapid test kits available

– Latex agglutination test based on antigen and antibody agglutination



• Streptococcus screening– Rules for accurate screening

• Use correct swab in taking throat cultures

• Always run positive and negative control along with actual test

• Read and understand directions before starting test



• Streptococcus screening– Rules for accurate screening

• Never use outdated kits and materials

• Observe all safety guidelines


Sensitivity Testing

• Often ordered on pathogenic organisms recovered from culturing process


Parasitology

• Becoming more common

• Geographical area determines types of parasites seen

• Examination methods


Parasitology

• Specimen collection– Use of wide-mouth containers with tight lid to prevent

leakage

– Strictly follow laboratory procedure

– Label specimen correctly

– Follow standard precautions and OSHA guidelines


Parasitology

• Common parasites– Enterobius vermicularis

• Pinworms

• Nematode

– Diagnosis of


Parasitology

• Common parasites– Trichomonas vaginalis

• Found five times more often in women than in men

• Transmitted sexually

• Recovery of trichomonad


Mycology

• Extensive field

• Candida has several species that cause yeast infections in body

• Dermatophytes

Copyright © 2010 Delmar, Cengage Learning. ALL RIGHTS RESERVED. Chapter 43 Basic Microbiology.

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