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

The Medical Assistant’s Role in the Microbiology Laboratory Use exact technique to avoid laboratory error Use sterile equipment Send culture to laboratory in reasonable amount of time

The Medical Assistant’s Role in the Microbiology Laboratory 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 Species 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 Media 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 Handling Microbiology 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

Safety When Handling Microbiology Specimens 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

Safety When Handling Microbiology Specimens 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

Safety When Handling Microbiology Specimens Specimen handling Check for leaks and contamination on containers Wear gloves Use appropriate container Handle all specimens as if contaminated

Safety When Handling Microbiology Specimens 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

Collection Procedures Common microbiology specimen sites Place in appropriate container Bring to laboratory Rejecting specimens

Collection Procedures 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

Collection Procedures 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

Specific Collection Requirements Throat Use sterile tongue depressor to hold patient’s tongue down Avoid swabbing sides of mouth and tongue

Specific Collection Requirements Wound Use of sterile needle or swab to aspirate pus-filled fluid from wound Use of anaerobic transport medium

Specific Collection Requirements Sputum Patient coughs deeply and expectorates into sterile container Should be morning specimen Use of special container

Specific Collection Requirements Click Here to play the video

Specific Collection Requirements Stool Ova and parasites Bacterial cultures Non-sterile containers Contamination of urine

Specific Collection Requirements Cerebrospinal fluid (CSF) Lumbar puncture Fluid dispersed in several departments of clinical laboratory Use of incubator Refrigeration can kill meningitis-causing bacteria

Specific Collection Requirements Blood Development of septicemia Collection of cultures Variety of collection devices available

Bacterial Shapes Cocci 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

Microscopic Examination of Bacteria 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

Microscopic Examination of Bacteria 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

Microscopic Examination of Bacteria Differential stain Identifies gram-positive and gram-negative bacteria Staphylococcus Streptococcus E. coli Proteus Morphological arrangement, shape, and gram-stain characteristic help identify bacteria

Microscopic Examination of Bacteria Acid-fast stain Specific stain Allows microscopic examination of acid-fast mycobacteria Use of heat or powerful dye Ziehl-Neelsen stain Kinyoun stain

Microscopic Examination of Bacteria Special techniques Used when flagella, spore, capsule, or nuclei of cells are present Tests without staining Wet slide preparation Hanging drop

Microscopic Examination of Bacteria 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

Microscopic Examination of Bacteria 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 Broth tubes store semisolid media 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

Microbiology Culture Inoculating the media After inoculating agar plate, turn upside down and place in proper environment for growth Broth tube inoculation Deep inoculation/slant

Microbiology Culture 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

Microbiology Culture Primary culture Read after media incubated 24–48 hours Observe for gross colony characteristics Size Shape Color Elevation

Microbiology Culture Primary culture Observe for gross colony characteristics Density Consistency Hemolytic versus nonhemolytic Odor Pigment production Use of bright, direct light

Microbiology Culture 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

Identification Systems 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

Identification Systems 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 Diagnosis of 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