Lab 4

How clean is clean? Do disinfectants sterilize? Are antiseptics germicidal? Are bacteriostatic and bactericidal synonyms? Does decontamination always eliminate pathogens? When should each method be used?

Decontamination includes all methods of microbial control.

• Decontamination procedures destroy or remove contaminants.
• In microbiology, the contaminants are any microorganisms present that are undesirable.
• The most resistant contaminants are bacterial endospores.
• Moderate resistance is common among protozoan cysts, some fungal sexual spores, and some viruses. Generally speaking, naked viruses are more resistant than enveloped viruses (you'd think it'd be the other way!). The most resistant viruses are the hepatitis B virus and the poliovirus.
• Vegetative bacteria (non-endospore-forming) that are moderately resistant are M. tuberculosis, S. aureus, and the Pseudomonas species.
• The least resistant contaminants include all other bacterial vegetative cells, ordinary fungal spores and hyphae, enveloped viruses, yeasts, and trophozoites.
• Decontamination methods involve physical agents (e.g., heat, radiation) and chemical agents (e.g., disinfectants, antiseptics).
• Decontamination methods' destructive effects are considered final when "the permanent loss of reproductive capability, even under optimum growth conditions," (Foundations in Microbiology, Talaro and Talaro) has occurred. This is the accepted microbiological definition of death.
• Today's lab will discuss three decontamination methods, sterilization, disinfection, and sanitization.

Sterilization destroys all microorganisms, including endospores. Recall that endospores are the most resistant form that bacteria can take.

• Moist heat under high pressure is generally used to sterilize.
• Autoclaves are able to achieve temperatures above that of boiling water to sterilize all objects that are not damaged by heat or moisture.

• Steam at a pressure of 15 psi has a temperature of 121^o C.
• Thirty minutes under these conditions will kill all organisms and their endospores.
• Quality assurance is important, and indicators such as "killit ampules" are used to assure that appropriate pressures and temperature were achieved.
• A sterilizing gas, such as ethylene oxide, can also be used.
• There are degrees of sterilization; "commercial sterilization" does not kill all endospores, although it kills C. botulinum spores. That's good enough for food.

Cleanliness is relative!

Disinfection destroys harmful microorganisms (pathogens).

• Disinfection is not the same as sterilization, because it does not destroy endospores.
• Disinfection destroys vegetative cells (non-endospore-forming).
• Disinfection involves the use of chemicals, ultraviolet radiation, boiling water, or steam.
• Disinfectants generally refer to the application of chemicals on inert objects (non-living).

• Disinfectants applied to living tissue are called antiseptics.
• Generally speaking, disinfectants are stronger than antiseptics (why?).
• How to tell whether the disinfectant really kills bugs dead or merely discourages them from growing and multiplying? Look at the suffix:
• -cide: This is a killer. Whatever preceded this suffix is dead after its use.
• -static: This maintains the status quo. Whatever preceded this suffix is not going to set up house and multiply, but it's not dead either, so reapplications are necessary to maintain acceptable standards of cleanliness.

Sanitization is any cleansing technique that mechanically removes microorganisms to reduce the level of contaminants to safe levels.

• Degerming is the mechanical removal of microbes. It does not kill, unless an antiseptic is used as the degermer. Non-antiseptic degermers move the bacteria around--hopefully away (so, pay attention to how you swab!). Alcohol swabs are examples.
• Sanitizers lower microbial counts to acceptable levels, thus reducing the risk of disease transmission. Soap is an example.

It's easy to stop the spread of infection, right?

• Just remove the source of infecting organisms...OR
• Remove the susceptible host...OR
• Remove the means of transmission (the bridge) between the microorganisms and the susceptible host...

Easier said than done?

In a hospital setting, the first two cannot be removed. Consequently, health care workers are charged with the very serious responsibility of ensuring that the bridge between source and prospective host is never completed.

Most hospitals have certain precautions in place that safeguard the health of both patients and staff. These include standard precautions and transmission-based precautions.

• Standard precautions are to be used for all patients, regardless of patient diagnosis.
• Standard precautions apply to blood and body fluids, secretions, excretions, non-intact skin, and mucous membranes.
• Standard precautions are designed to reduce the risk of transmission of microorganisms from both recognized and unrecognized sources of infection.
• Standard precautions require the use of gloves when handling blood or body fluids or non-intact skin.
• Standard precautions require prompt and thorough handwashing between patients and/or patient secretion contacts (Always wash hands after removing gloves!).
• Standard precautions require mask/eye protection/face shields and gown to be worn during procedures likely to generate splashes/sprays of blood/body fluid.

• Transmission-based precautions are used in conjunction with standard precautions.
• Transmission-based precautions are used when a patient is actually known or suspected to be infected with a highly transmissible or epidemiologically important pathogen.
• There are three kinds of transmission-based precautions, airborne, droplet, and contact precautions, which may be combined together for diseases that are spread by more than one route.

• Standard precautions observed.
• Patient is placed in a negative air pressure room having 6-12 air changes/hour and monitored high-efficiency filtration of the room air.
• Particulate filter respirator capable of filtering out particles as small as 0.3 micrometers (e.g., PFR 95) is worn before entering patient room and discarded after exiting patient room.
• If patient is to be transported, place surgical mask on patient, if possible.
• Airborne precautions are taken when patients are known or suspected to have serious illnesses transmitted by airborne droplet nuclei, such as measles (rubeola), varicella (chickenpox and shingles), and tuberculosis.
• Airborne transmission is not the same as droplet transmission!

• Standard precautions observed.
• Surgical mask worn when working in close proximity of patient.
• If patient is to be transported, place surgical mask on patient, if possible.
• Droplet precautions are taken when patients are known or suspected to have serious illnesses caused by microorganisms transmitted through the droplets that are sprayed when a patient talks, coughs, or sneezes. Examples of these organisms are Haemophilus influenza, Neisseria meningitidis, and those viruses causing influenza, mumps, and rubella. These droplets do not remain suspended in the air and are generally not transmissible beyond three feet.
• Droplet transmission is not the same as airborne transmission!

• Standard precautions observed.
• Gloves and gown worn before entering patient room and discarded before exiting patient room.
• Contact precautions are used when a patient is known or suspected to be colonized or infected by an epidemiologically important microorganism that is transmitted through direct or indirect contact.
• Direct contact involves skin to skin contact and physical transfer of the microorganism from the infected individual to a susceptible host.
• Indirect contact occurs when an intermediate object, termed a "fomite," such as tape, a thermometer, a ventilator, a doorknob, or other inanimate object, has been contaminated with the microorganism. Transmission occurs when a susceptible host comes in contact with the contaminated object.
• Epidemiologically important microorganisms transmitted by contact include all multidrug resistant organisms such as methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus (VRE) and organisms such as Clostridium dificile.

Today, we will be preparing for next week's lab in which we will determine the efficacy of various disinfectants. The disinfectants to be used include:

• Lysol, at recommended concentration
• Clorox, at recommended concentration (5.25%)
• Isopropanol, at recommended concentration (70%)
• Betadine, undiluted
• 3% Hydrogen peroxide, at recommended concentration (10%)
• Ethanol, at recommended concentration (70%)
• Scope-quaternary ammonium, undiluted

You will be provided with one of the above disinfectants and a broth culture of bacteria. The purpose of this activity is to determine the lowest dilution of disinfectant that inhibits the growth of bacteria.

1. You will use the disinfectant to make an undiluted 0.1 mL inoculation of nutrient broth for your negative control.
2. Your positive control will consist of a 0.1 mL inoculation of bacteria to nutrient broth that does not contain any disinfectant.
3. The rest of your nutrient broths will be inoculated with 0.1 mL of bacteria and serial dilutions of the disinfectant.
4. All tubes will be incubated for 24-72 hours, depending on the growth of the positive control, and then refrigerated until the following week's lab.

In addition, you will determine the time required for the bactericidal effects of disinfectants at recommended concentrations to become evident. You will be given a broth culture of bacteria, three plates, and one tube containing disinfectant at the recommended concentration.

1. Each plate will be used for two streaking treatments, so draw an imaginary line down the middle of the petri plate.
2. Each plate will need to be labeled (on the agar side) appropriately.
3. The negative control will consist of a loopful of disinfectant streaked across one half of the petri plate.
4. The positive control will consist of a loopful of the bacterial broth streaked across one half of the petri plate.
5. Add 0.1 mL of bacteria into the tube containing disinfectant (after you have completed step 3 above).
6. At 2, 5, 10, and 20 minutes, streak the petri plate with a loopful of the inoculated disinfectant. Be sure to streak on the appropriately labeled side of the petri dish!
7. The three plates will be incubated for 24-72 hours, depending on the growth of the positive control, and then refrigerated until the following week's lab.

The following week, you will determine whether growth occurred in your broths and plates. Based on what you observe, you will be able to determine three things:

1. The difference in disinfectant efficacy based on concentration of disinfectant.
2. The difference in disinfectant efficacy based on time of contact with disinfectant.
3. The difference in disinfectant efficacy base on Gram positive or Gram negative bacterial status.

Take Out Food for the Brain:

An interesting trip through infection control history [Thanks to Steve Rose, BSMT(ASCP), SM]:

Quarantine: The practice of quarantine began during the fourteenth century to protect coastal cities from visitations of epidemics of plague on arriving ships. Ships were forced to sit at anchor for forty days before disembarkation was permitted. Travelers arriving by land were forced to stay in huts outside the village for forty days. The Latin word for forty is quaresma; thus began the concept of quarantine.

As Europe suffered through a plague that wiped out half the continent's population and took 300 years to restore, ultimately causing the disappearance of Latin as a spoken language, people blamed infectious diseases on poisonous vapors, sin, an angry God, and foreigners.

Microorganisms were discovered to be the cause of many serious diseases, such as cholera and tuberculosis. The Germ Theory and Koch's Postulates replaced divine retribution as the cause of infectious diseases. Populations shifted from country to city.

In the United States, industrialization and immigration led to overcrowding and deplorable housing conditions. Public water supplies were inadequate and waste-disposal systems were non-existent. There were outbreaks of cholera, dysentery, tuberculosis, typhoid fever, influenza, yellow fever, and malaria.

When the United States was first established, not much thought was given to the importation of infectious diseases. It was the yellow fever epidemics that led to the passage of Federal Quarantine Legislation by Congress in 1878. This followed the first published recommendation for placing patients with infectious diseases in separate facilities, called Infectious Disease Hospitals.

Nursing textbooks described aseptic procedures, and infectious patients were segregated by disease in wards or floors. Yet non-aseptic techniques continued to claim the lives of almost half of all women dying of pregnancy complications (CDC reports that 40% of maternal deaths were due to sepsis). This continued through the early 1920s.

The cubicle system of isolation was set up. This consisted of multiple bed wards and nurses practicing barrier methods of infection control. Barrier nursing included the use of separate gowns between patients, the use of antiseptic for handwashing, and the use of disinfectants on objects touched by patients.

Alexander Fleming, who was later awarded the Nobel Prize in Physiology and Medicine for his work, discovered penicillin.

Penicillin was produced in substantial quantities and used first by the US military to treat sick and wounded soldiers. The first US civilian whose life was saved by penicillin was a 33 year-old woman who had been hospitalized for over a month with a life-threatening streptococcal infection. Her temperature rose to 107^oF, and she was close to death before her doctors, in desperation, injected her with a tiny amount of an "obscure experimental drug called penicillin." She recovered, married, raised a family, met Sir Alexander Fleming, and died last June at the age of 90.

US Infectious Disease Hospitals began to close, except for Tuberculosis Sanitariums. Patients were still losing limbs and dying because of staphylococcal infections.

Tuberculosis sanitariums began to close.

Patients moved from wards into single-patient isolation rooms or regular single and multiple patient rooms.

CDC introduces 7 isolation categories for grouping and dealing with infectious disease patients.

The previous infectious disease category system is revised. Infection Control manuals are developed at hospitals across the nation.

Hospitals are experiencing new endemic and epidemic nosocomial infection problems, including multidrug resistant organisms and newly recognized pathogens.

CDC revises isolation categories again.

HIV epidemic in full force. Isolation practices dramatically altered and Universal Precautions are introduced to protect hospital personnel.

Body Substance Isolation protocols urged by a Seattle hospital.

CDC posts recommendations for patients suspected to have hemorrhagic fever.

Occupational Safety and Health Administration (OSHA) enters healthcare scene.

Many issues, some political, make infection control almost oxymoronic: multidrug resistance, rise in tuberculosis, new emerging diseases, shorter hospital stays, increased care of the sick in the community, decrease in public health staff, overuse of antibiotics. Public attention is riveted on emergence of Hantavirus, Ebola, and drug-resistant organisms--up until now, it was thought that there was a magic bullet for every infectious disease.

OSHA Bloodborne Pathogen Regulations instituted.

CDC revises Isolation Guidelines, ultimately resulting in Standard Precautions (which were a combination of Universal Precautions and Body Substance Isolation) and Transmission Based Precautions

Where do we go from here? You tell me.

According to the CDC, airplanes have replaced ships as major vehicles of international disease spread.