Lecture 1 Safety and Basic Microbiology Techniques

I. Microbiology General Issues

A. Microbiology Techniques

1.    Cultivation

a)    Culture

b)   Biochemical  examination – metabolic by-products

c)    Sensitivity

2.    Direct Observation

a)    Microscopic

                                                             i.      Light microscope

                                                           ii.      Fluorescent abs & UV microscope

3.    Direct detection through specific products

a)    Immunologic techniques

b)   Molecular techniques - DNA

4.    Serology – antibody or antigen detection

 B. Transportation of Specimens

1.    Collection, place, timing

2.    Transport and transport media

3.    Preservation

4.    Storage

5.    Rejecting Unacceptable specimens

         C. Culturing

1.    Media

a)    Enriched, selective specialized

b)   Broth, solid

2.    Incubation

a)    Temperature

b)   Aerobic vs anaerobic

 II. Lab Safety


1. Prevention of infection with laboratory cultures.
2. Prevention of harm from lab equipment.
3. Proper containment of hazardous waste.
4. Manage safety breaches by recording and correcting any unsafe practices.
5. Universal precautions
6. Material Safety Data Sheet - MSDS

A. Microbes used in the Microbiology lab may cause harm if they are introduced into a susceptible person.

1.    Agent safety assessment.

Biosafety levels: 1, 2, 3, 4.

2.    Common agents the microbiology lab

     3.    Proper treatment and protocol

     4.    Knowledge of risk associated with labs and biosafety containment .

 B. Safety equipment

1.    Laminar flow hood

2.    UV light

3.    Incinerators and flammable reagents

4.    Microscope and electrical outlet

5.    Autoclave

6.    Sinks

7.    Hypodermics and other sharp objects

8.    Slide warmer, incubator

9.    Reagents

10.                       Stains

11.                       centrifuge

C. Safe disposal of biohazards requires careful training and waste streaming.

1.    Biohazard table – sharps, glass, plates

2.    Biohazard bags

     3.    Minimize contact, timing and sterilization

     4.    Spills

D. Recording safety breaches –

1.    Alert supervisor immediately

2.    Document and consult MSDS

3.    Assess cause and correct procedure or practice

 E. Universal Precautions

1.    Barriers – PPG – gloves, masks, labcoats

Perspectives in Disease Prevention and Health Promotion Update: Universal Precautions for Prevention of Transmission of Human Immunodeficiency Virus, Hepatitis B Virus, and Other Bloodborne Pathogens in Health-Care Setting http://www.cdc.gov/mmwr/preview/mmwrhtml/00000039.htm

The risk of nosocomial transmission of HIV, HBV, and other bloodborne pathogens can be minimized if health-care workers use the following general guidelines:**

  1. Take care to prevent injuries when using needles, scalpels, and other sharp instruments or devices; when handling sharp instruments after procedures; when cleaning used instruments; and when disposing of used needles. Do not recap used needles by hand; do not remove used needles from disposable syringes by hand; and do not bend, break, or otherwise manipulate used needles by hand. Place used disposable syringes and needles, scalpel blades, and other sharp items in puncture-resistant containers for disposal. Locate the puncture-resistant containers as close to the use area as is practical.
  2. Use protective barriers to prevent exposure to blood, body fluids containing visible blood, and other fluids to which universal precautions apply. The type of protective barrier(s) should be appropriate for the procedure being performed and the type of exposure anticipated.
  3. Immediately and thoroughly wash hands and other skin surfaces that are contaminated with blood, body fluids containing visible blood, or other body fluids to which universal precautions apply. Glove Use for Phlebotomy

    Gloves should reduce the incidence of blood contamination of hands during phlebotomy (drawing blood samples), but they cannot prevent penetrating injuries caused by needles or other sharp instruments. The likelihood of hand contamination with blood containing HIV, HBV, or other bloodborne pathogens during phlebotomy depends on several factors: 1) the skill and technique of the health-care worker, 2) the frequency with which the health-care worker performs the procedure (other factors being equal, the cumulative risk of blood exposure is higher for a health-care worker who performs more procedures), 3) whether the procedure occurs in a routine or emergency situation (where blood contact may be more likely), and 4) the prevalence of infection with bloodborne pathogens in the patient population. The likelihood of infection after skin exposure to blood containing HIV or HBV will depend on the concentration of virus (viral concentration is much higher for hepatitis B than for HIV), the duration of contact, the presence of skin lesions on the hands of the health-care worker, and -- for HBV -- the immune status of the health-care worker. Although not accurately quantified, the risk of HIV infection following intact skin contact with infective blood is certainly much less than the 0.5% risk following percutaneous needlestick exposures (5). In universal precautions, all blood is assumed to be potentially infective for bloodborne pathogens, but in certain settings (e.g., volunteer blood-donation centers) the prevalence of infection with some bloodborne pathogens (e.g., HIV, HBV) is known to be very low. Some institutions have relaxed recommendations for using gloves for phlebotomy procedures by skilled phlebotomists in settings where the prevalence of bloodborne pathogens is known to be very low.

    Institutions that judge that routine gloving for all phlebotomies is not necessary should periodically reevaluate their policy. Gloves should always be available to health-care workers who wish to use them for phlebotomy. In addition, the following general guidelines apply:

  4. Use gloves for performing phlebotomy when the health-care worker has cuts, scratches, or other breaks in his/her skin.
  5. Use gloves in situations where the health-care worker judges that hand contamination with blood may occur, for example, when performing phlebotomy on an uncooperative patient.
  6. Use gloves for performing finger and/or heel sticks on infants and children.
  7. Use gloves when persons are receiving training in phlebotomy. Selection of Gloves

    The Center for Devices and Radiological Health, FDA, has responsibility for regulating the medical glove industry. Medical gloves include those marketed as sterile surgical or nonsterile examination gloves made of vinyl or latex. General purpose utility ("rubber") gloves are also used in the health-care setting, but they are not regulated by FDA since they are not promoted for medical use. There are no reported differences in barrier effectiveness between intact latex and intact vinyl used to manufacture gloves. Thus, the type of gloves selected should be appropriate for the task being performed.

    The following general guidelines are recommended:

  8. Use sterile gloves for procedures involving contact with normally sterile areas of the body.
  9. Use examination gloves for procedures involving contact with mucous membranes, unless otherwise indicated, and for other patient care or diagnostic procedures that do not require the use of sterile gloves.
  10. Change gloves between patient contacts.
  11. Do not wash or disinfect surgical or examination gloves for reuse. Washing with surfactants may cause "wicking," i.e., the enhanced penetration of liquids through undetected holes in the glove. Disinfecting agents may cause deterioration.
  12. Use general-purpose utility gloves (e.g., rubber household gloves) for housekeeping chores involving potential blood contact and for instrument cleaning and decontamination procedures. Utility gloves may be decontaminated and reused but should be discarded if they are peeling, cracked, or discolored, or if they have punctures, tears, or other evidence of deterioration. Waste Management

2.    Fluids that transmit blood borne pathogens

3.    Isolation

Table 1
Synopsis of Types of Precautions and Patients Requiring the Precautions*

Standard Precautions
    Use Standard Precautions for the care of all patients
Airborne Precautions
    In addition to Standard Precautions, use Airborne Precautions for patients known or suspected to have serious illnesses transmitted by airborne droplet nuclei. Examples of such illnesses include:
    Varicella (including disseminated zoster)†
Droplet Precautions
    In addition to Standard Precautions, use Droplet Precautions for patients known or suspected to have serious illnesses transmitted by large particle droplets. Examples of such illnesses include:
    Invasive Haemophilus influenzae type b disease, including meningitis, pneumonia, epiglottitis, and sepsis
    Invasive Neisseria meningitidis disease, including meningitis, pneumonia, and sepsis
    Other serious bacterial respiratory infections spread by droplet transmission, including:
    Diphtheria (pharyngeal)
    Mycoplasma pneumonia
    Pneumonic plague
    Streptococcal (group A) pharyngitis, pneumonia, or scarlet fever in infants and young children
    Serious viral infections spread by droplet transmission, including:
    Parvovirus B19
Contact Precautions
    In addition to Standard Precautions, use Contact Precautions for patients known or suspected to have serious illnesses easily transmitted by direct patient contact or by contact with items in the patient's environment. Examples of such illnesses include:
    Gastrointestinal, respiratory, skin, or wound infections or colonization with multidrug-resistant bacteria judged by the infection control program, based on current state, regional, or national recommendations, to be of special clinical and epidemiologic significance
    Enteric infections with a low infectious dose or prolonged environmental survival, including:
    Clostridium difficile
    For diapered or incontinent patients: enterohemorrhagic Escherichia coli O157:H7, Shigella, hepatitis A, or rotavirus
    Respiratory syncytial virus, parainfluenza virus, or enteroviral infections in infants and young children
    Skin infections that are highly contagious or that may occur on dry skin, including:
    Diphtheria (cutaneous)
    Herpes simplex virus (neonatal or mucocutaneous)
    Major (noncontained) abscesses, cellulitis, or decubiti
    Staphylococcal furunculosis in infants and young children
    Zoster (disseminated or in the immunocompromised host)†
    Viral/hemorrhagic conjunctivitis
    Viral hemorrhagic infections (Ebola, Lassa, or Marburg)*

* See Appendix A for a complete listing of infections requiring precautions, including appropriate footnotes.
† Certain infections require more than one type of precaution.
‡ See CDC "Guidelines for Preventing the Transmission of Tuberculosis in Health-Care Facilities."(23)


Updated: February 18, 1997



F. Material Safety Data Sheet – MSDS

1.    Location

2.    Information