4 MICROBIOLOGY

• Microbiology


  The study of living organisms that are too small to be seen by the naked eye
• Biological Safety Cabinet

  • Contains a HEPA (High Efficiency Particulate Air) filter that removes dust, pollen, bacteria, and any airborne particles with at least a size of 0.3microns
  • Allows room air to pass into cabinet and around the area and material within, sterilizing only the air to be exhausted
  • Consists of negative pressure
  • Operated in open front
• Classes of Biosafety Cabinet

  • Class I
  • Class II
  • Class IIA
  • Class IIB
• Microbes

  Microscopic organisms
• Classifications of Microbes

  • Acellular Microorganisms (Prions, Viruses)
  • Cellular Microorganisms (Prokaryotes - Archaea, Bacteria; Eukaryotes - Algae, Fungi, Protozoa)

  
• Subdivisions of Microbes

  • Virus
  • Bacteria
  • Fungi
  • Parasite
• Virus

  • Very simple microbes, consisting of nucleic acid, a few proteins, and in some, a lipid envelope
  • Completely dependent on the cells they infect for their survival and replication
• Bacteria


  • Has both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), a metabolic machinery for self-replication, and a complex cell wall structure
  • Regarded as prokaryotic or simple unicellular organisms
  • Reproduce by asexual division
• Subdivisions of Fungi

  • Single-celled organisms called yeasts
  • Multi-celled organisms called molds
  • Medically important members existing in both forms called dimorphic fungi
• Subdivisions of Parasites

  • Single-celled organisms called protozoa
  • Multi-celled organisms called worms and bugs

  
• Bacteria

  • Prokaryotic unicellular organisms that lack a true nucleus and nuclear membrane
  • Single, closed, circular chromosomes of double-stranded DNA called NUCLEOID
  • PLASMIDS: small circular molecules of extrachromosomal circular DNA
  • May either be Gram Positive (Violet) or Gram Negative (pink or reddish pink)
  • BINARY FISSION: asexual reproduction of bacteria
  • Bacteria size: range from 0.2 to 2um in diameter and 1 to 6um in length

  
• Gram Stain


  • Principal stain used for microscopic examination of bacteria
  • First devised by Hans Christian Gram during the late 19th century
  • Divides most bacterial species into two large groups: Gram-positive (take up the basic dye, crystal violet) and Gram-negative (allow crystal violet dye to wash out easily with decolorizer alcohol or acetone)
• Differences between Gram Positive and Gram Negative Bacteria

  • Gram Positive: Thick cell wall (peptidoglycan), Absence of lipopolysaccharide, Purple or Violet result
  • Gram Negative: Thin cell wall (peptidoglycan), Presence of lipopolysaccharide, Red or Pink result

  
• Steps in Gram Staining

  1. Fix sample into slide

  2. Use crystal violet dye as primary dye

  3. Use iodine as mordant to fix dye on bacteria

  4. Use alcohol or acetone as decolorizing agent

  5. Counterstain with safranin
• Four Basic Morphological Types of Bacteria

  • Cocci: spherical-shaped cells
  • Bacilli: rod-shaped cells
  • Spirilla: spiral shaped cells
  • Vibrios: comma-shaped cells

  
• Two Types of Bacterial Metabolism

  • Fermentation
  • Respiration
• Fermentation


  Process by which bacteria catabolize carbohydrates to produce energy
• Respiration


  • Process of bacterial energy generation rather than fermentation
  • Can be aerobic (occurs in the presence of oxygen) or anaerobic (occurs in the absence of oxygen)
• Universal Precautions (UP) are followed throughout the collection and handling process
• Persons collecting or handling specimens should wear gloves and a laboratory gown
• Eye protection should be worn if splashing
• Accidents or injuries (needle prick) must be reported immediately
• General Guidelines for Specimen Collection

  • Specimen should be from the infection site (within the wound) and not contaminated by the surrounding area
  • Specimen should be collected before antimicrobials are administered (antibiotics)
  • Appropriate collection devices and containers should be used and must be STERILE! ASEPTIC TECHNIQUE is required
  • Specimen container should be labeled with patient's identification, the date and time of collection and the source of specimen
• Body Sites for Specimen Collection

  • Throat (swabs)
  • Sputum
  • Stool
  • Urine
  • Blood
  • Cerebrospinal fluid
  • Genital tract
• Throat Swab Collection

  1. Tongue should be depressed before swabbing between the tonsillar pillars and behind the uvula

  2. The cheek, tongue, and teeth should NOT be touched
• Sputum Collection

  1. Expectorated specimens from deep cough should be collected into a sterile specimen cup

  2. Early morning specimens are the best
• Stool Collection


  Should never be taken from the toilet and should not be contaminated with urine
• Urine Collection

  1. Midstream clean-catch is the most common collection method

  2. Culture for catheterized urine specimens usually have less contaminating bacterial flora
• Blood Collection

  1. 2-3 cultures should be collected at random times during 24-hour period

  2. Venipuncture

  3. Skin is disinfected with 70% alcohol, followed by Iodine

  4. ADULT: 20-30mL of blood per culture is collected

  5. INFANTS: 1-5mL of blood per culture is collected
• Cerebrospinal Fluid Collection

  1. Should be collected aseptically by PHYSICIAN

  2. Should be processed immediately and not exposed to heat or refrigeration
• Genital Tract Collection

  1. MEN (penile discharge): Exudates may be expressed from the urethral orifice or a small-diameter swab inserted 3-4 cm into the urethra

  2. WOMEN (vaginal discharge): A sterile swab is inserted into the cervix with an aid of speculum, swab is rotated and allowed to remain for a few seconds

  3. Samples taken from genital tract should not be refrigerated
• Anaerobic specimens must be transported in an anaerobic system
• Swab samples are not allowed to dry out
• Most specimen can be held at 2°C to 8°C [refrigerator temperature] if transport cannot occur immediately EXCEPT temperature-sensitive organisms, blood culture bottles, and CSF
• Culture Media


  An artificial preparation in the laboratory which contains the basic foundations of nutrients
• Types of Culture Media

  • Nutritive Media
  • Differential Media
  • Selective Media
  • Backup Broth
  • Specialized Media
• Nutritive Media


  Support the growth of a wide range of microorganisms and are considered non selective
• Differential Media


  Microorganisms that can be distinguished on the basis if certain growth characteristics evident on the medium
• Selective Media


  Support the growth of one group of organisms, but not another, by adding antimicrobials, dyes, or alcohol to a particular medium
• Backup Broth


  Also called supplemental or enrichment broth, for detection of small numbers of organisms present