Lab 5

Objectives:

Reading:

1. Disinfection: Time, Concentration and Gram Reaction

1. Handouts

Time, Concentration, and Gram Reaction

Which variables contribute to the selection of a disinfectant and its ability to eliminate pathogens?

  1. Concentration of the disinfectant
  2. Time of contact with the disinfectant
  3. Gram reaction of the organism being disinfected
  4. Organic contamination of the disinfectant
  5. Object being disinfected (inanimate vs. animate)
  6. Cost of disinfectant

Today's lab will focus on the relationship of contact time, disinfectant concentration, and Gram reaction.

There are two parts to the lab, and each part is performed using both a Gram negative and a Gram positive bacterial broth culture.

The disinfectants to be used include:

 

 

Test of Effect of Concentration of Disinfectant on Anti-Microbial Capabilities:

Materials:

Broth culture containing Staphylococcus epidermidis

Broth culture containing Escherichia coli

One tube of undiluted disinfectant

Three tubes of nutrient broth, each containing 2.7 mL of nutrient broth

Two tubes of nutrient broth for positive and negative controls

Wax pencil

Pipette

 

Instructions:

  1. Mark the three tubes with their dilution (10-1, 10-2, etc.) and an identifier such as initials, group name, etc.

  2. Using the pipette, measure 0.3 mL of undiluted disinfectant into the first of the three tubes. This represents a 1:10 dilution of the disinfectant (0.3/3.0).

  3. Mix contents of this first dilution, and using the pipette, transfer 0.3 mL of this dilution into the second tube, labeled 10-2.

  4. Mix and continue serially diluting.

  5. Once the third tube has been diluted, transfer 0.3 mL of its contents into the BioHazard Discard Can on the counter top (just to keep all the volumes the same).

  6. Transfer 0.1 mL of undiluted disinfectant into the tube labeled 'negative control.' This tube will contain 2.7 mL of nutrient broth and 0.1 mL of undiluted disinfectant only.

  7. Transfer 0.1 mL of bacterial broth culture into each of the three tubes. Pay attention to which bacterial broth you are supposed to be using!

  8. Transfer 0.1 mL of bacterial broth culture into the tube labeled 'positive control.' This tube will contain 2.7 mL of nutrient broth and 0.1 mL of bacterial broth only.

  9. Transfer 0.1 mL of bacterial broth culture into the tube containing the undiluted disinfectant.

  10. The six tubes will be incubated at 37oC. for 24-72 hours (depending on the growth of the positive control) and then refrigerated until next week's lab. At that time, you will complete a table that describes the growth patterns you observe.

 

 

 

Test of Effect of Contact Time of Disinfectant on Anti-Microbial Capabilities:

Materials:

Broth culture containing Staphylococcus epidermidis

Broth culture containing Escherichia coli

One tube of disinfectant at recommended concentration

Three petri dishes

Wax pencil

Inoculating loop

Bunsen burner

 

Instructions:

  1. Mark the plates. Be sure to include an identifier.

    2.  

  2. Sterilize your inoculating loop (allowing it to cool) and insert it into the tube of disinfectant, using aseptic technique. Streak the half of the petri dish that is labeled 'negative.' Do not streak for isolation; simply streak in a zigzag fashion across the lower half of the dish. Sterilize your loop when done.
  3. Now add 0.1 mL of bacterial broth culture into the tube of disinfectant. Pay attention to which bacterial broth you are supposed to be using! This is "zero time." Begin keeping track of your time.
  4. After two minutes, sterilize your loop and streak a loopful of the bacterial broth/disinfectant mixture onto the side of the petri dish labeled '2.' Sterilize your loop.
  5. After five minutes, repeat, streaking onto the side of the petri dish labeled '5.'
  6. After ten minutes, repeat, streaking onto the side of the petri dish labeled '10.'
  7. After 20 minutes, repeat, streaking onto the side of the petri dish labeled '20.'
  8. At some point, you need to remember to streak your positive control, using the bacterial broth (NOT the bacterial broth/disinfectant mixture).
  9. 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. At that time, you will complete a table that describes the growth patterns you observe.

 

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:

The use of antibiotics is reported to lead to bacterial resistance to antibiotics. This is a challenging statement that should give any conscientious health care worker pause.

Case in point:

Staphylococcus bacteria are transmitted by skin to skin contact. When they gain access into the body through open wounds, they can become pathogenic. Staphylococcus aureus infect the skin, soft tissue, bones, and joints. According to the Centers for Disease Control and Prevention (CDC), these infections are responsible for 13% of the over two million annual nosocomial infections in the United States.

Until very recently, all methicillin-resistant Staphylococcus aureus (MRSA), the most resistant of the Staphylococcal pathogens, were known to be sensitive to vancomycin. Vancomycin is bactericidal against many gram-positive and some gram-negative cocci. However, it is toxic and should be used with caution, especially among patients whose renal function is insufficient. Consequently, vancomycin is considered to be a drug of last resort, and blood levels are frequently monitored in order to prevent potential toxic effects.

In April, 1999, the CDC received the news that all epidemiologists had been expecting but hoping was still a long way in coming: the first confirmed death of a patient with MRSA in her heart valves whose infection did not respond to vancomycin. The following November, three people were reported to have died in Hong Kong from a similar strain.

Although researchers are hard at work in their efforts to develop new antibiotics, at the present time, there is no available antibiotic that can eliminate MRSA that are also resistant to vancomycin.

Is it irresponsible and alarmist of medical experts to declare that the use of antibiotics actually leads to bacterial resistance to antibiotics? You tell me.


 





Take Home Thought

WHAT DOESN'T KILL THEM MAKES THEM STRONGER










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