Antibiotics / Disinfectants

Notes:

When Pasteur disproved the concept of spontaneous generation the control of the growth of
microorganisms became of great interest. There are two major groups of chemicals that work
either by inhibiting cell growth or by killing the cells. Synthetic or human-made chemicals that
control microbial growth are called disinfectants and antiseptics. Chemicals produced naturally
from bacteria, fungi, or other microorganisms are called antibiotics. Some antibiotics are
synthetic or modified versions of natural types. Microbicidal or bacteriocidal means that these
agents kill the microorganism, while microbiostatic or bacteriostatic chemicals only inhibit the
growth of the microorganism. Another set of related terms are cytocidal and cytostatic (“cyto”
meaning cell).

Disinfectants
Disinfectants are synthetic antimicrobial agents that kill or prevent the growth of microorganisms
and should only be applied to inanimate objects. Some examples of disinfectants are phenol,
chlorine, and alcohol. Antiseptics are synthetic antimicrobial agents that can be safely applied
to tissues such as skin. An example of an antiseptic is iodine.

Phenol is one of the best chemical disinfectants. It is extremely irritating to skin and mucous
membranes. Derivatives of phenol called phenolics are more commonly used today. One
example of a phenolic is cresol, which is used in Lysol. Phenol and phenolics work by injuring
plasma membranes, inactivating enzymes and denaturing proteins.

Chlorine is used to control the growth of microorganisms usually found in water. This is done by
adding Hypochlourous acid (HOCl), bleach, to the water. Chlorine is a strong oxidizing agent; it
inactivates enzymes and denatures proteins.

Alcohol kills bacteria and is very effective in killing viruses as well. Alcohol will denature
proteins, disrupt membranes and dissolve many lipids including those found in viral envelopes.

In this lab we will be determining the effectiveness of some chemical disinfectants used in
hospitals or homes as antimicrobial agents.

Antibiotics
Antibiotics are chemicals that inhibit growth or kill microorganisms. Usually they are secondary
metabolites or by-products that are secreted out of the cell. It is thought that microbes produce
these compounds to kill off other competing organisms thereby improving the producer’s
chances of survival by increasing available resources; a form of biological warfare.

When treating disease, health care providers want to choose the best antibiotic. This is not as
easy as it sounds. First, the patient must not be allergic to the antibiotic, and second the
antibiotic must be effective in killing the microorganism or injuring it enough so that the immune
system can kill it. Today antibiotic resistant bacteria are becoming increasingly prevalent. One
way to reduce the risk of antibiotic resistance is to reduce the use of antibiotics for non-bacterial
infections. Antibiotics do not affect viruses.

Antibiotics can act in many ways, and some kill while others just inhibit growth or injure the
bacteria. Antibiotics such as penicillin and its derivatives can affect cell wall synthesis. Another
site of attack is the cell membrane. Tetracycline and streptomycin affect protein synthesis while
nalidixic acid interferes with RNA and DNA synthesis and replication.

Antibiotics fall into two categories. Broad-spectrum antibiotics can kill a wide range of
bacteria including both gram-positive and gram-negative species. Narrow spectrum antibiotics
can only kill a select few species of bacteria.

The first antibiotic discovered was penicillin. Alexander Fleming discovered penicillin when a
contaminating fungus stopped the bacterial growth on one of his plates. Penicillin was a
breakthrough discovery because it killed the bacteria with little or no side effects to the host,
although some individuals are allergic to this antibiotic. The search for new antibiotics has
undergone a recent revival in response to the spread of antibiotic resistant pathogenic bacteria.

One method used to determine antibiotic sensitivity is the Kirby-Bauer method. Bacteria are
mixed with soft agar and poured into the plate. Disks with known concentrations of antibiotic
are placed on the surface of the plate and the plates are incubated. Following incubation, the
bacteria will have grown forming visible turbidity in (or on) the plate. The size of the zone of
clearing around a disk where the bacteria have not grown, determines the sensitivity of the
bacteria to an antibiotic. Many factors can influence the zone of inhibition. 1) size of inoculum,
2) distribution of the inoculum, 3) incubation period, 4) depth of agar, 5) diffusion rate of the
antibiotic, 6) concentration of the antibiotic and 7) the growth rate of the bacteria.

In this lab, we will be using three antibiotics: ampicillin, streptomycin and tetracycline.
Ampicillin is a penicillin derivative classified as a broad-spectrum bacteriocidal antibiotic. It
works by inhibiting cell wall synthesis through inhibition of the enzyme transpeptidase.
Transpeptidase inserts and crosslinks newly formed peptidoglycan molecules into holes made in
the existing cell wall by a second enzyme, autolysin. If transpeptidase is inhibited, the
peptidoglycan molecules cannot be cross-linked, and the cell begins to leak nutrients, enzymes
etc. If there is a difference in osmotic pressure from the inside to the outside of the cell, lysis can
occur because the cell wall cannot hold the cell together.

Ampicillin is not effective against all bacteria or microorganisms. Some bacteria are resistant to
penicillin and its derivatives. These bacteria carry resistance genes on plasmids or phage that
have infected the cell. These genes produce β-lactamases, which destroy penicillin and its
derivatives by destroying the β-lactam ring (the reactive part of the antibiotic). Archaea are not
sensitive to ampicillin because they do not have peptidoglycan in their cell walls. Eukaryotes
either do not have a cell wall or the cell wall does not contain peptidoglycan.

Streptomycin is another broad-spectrum bactericidal antibiotic. This type of antibiotic inhibits

protein synthesis by binding to the 30S subunit of the bacterial ribosome. This causes
misreading of the mRNA, which in turn will cause incorrect proteins to be made, or in some
cases no protein at all. Since the bacteria cannot make the correct proteins, the cell dies.

Tetracycline is also broad-spectrum but it is bacteriostatic. This class of antibiotics also inhibits
protein synthesis. Tetracycline also binds to the 30S subunit of the bacterial ribosome,
specifically at the codon recognition site. This blocks amino acids from being added to a
peptide. If no amino acids are added to a peptide then no proteins can be made and the cell dies.
However, tetracyclines do not permanently bind, therefore one can remove the antibiotic by
dilution and the bacteria will begin to grow again. This antibiotic injures the cell, which gives
our immune system time to remove the bacteria from the body.

Hand Washing and Environmental Sampling

Good hand washing techniques and hygiene practices can prevent pathogenic bacteria from
causing an infection. In hospitals, hand washing is very important to reduce to spread of
pathogens and nosocomial infections. A nosocomial infection is one that is acquired in a
hospital. One commonly occurring nosocomial infection is a Staphylococcus infection acquired
with surgery.

Microorganisms are normally present in the human body but do not cause disease. Most animals
and humans have normal flora present in the gut such as colonies of Escherichia coli.
Microorganisms that are present for a short time without causing disease are transient flora.
For example, when you brush your teeth, transient flora may occur but disappear within a couple
of hours because they are removed by the immune system. A pathogen is a microorganism that
normally causes disease. Opportunistic pathogens are microorganisms that usually do not
cause disease; however, if the situation becomes right, the microorganism causes a disease. Most
nosocomial infections are caused by opportunistic pathogens.
Homework:

Disinfectants

1. What is the difference between microbicidal and microbiostatic?

2. What physical factors can influence the activity of a disinfectant?
3. Why do microorganisms differ in their response to disinfectants?

Antibiotics

1. How can you determine whether the zone of inhibition is due to death of a
bacterium or to inhibition of growth?
2. In which growth phase is a bacterium most sensitive to an antibiotic? Why?
3. What are some practices that would cause more bacterial strains to become resistant
to antibiotics?

Hand washing

1. Why could you see an increase in bacterial growth after hand washing?
2. Since most normal flora are not harmful, why are they removed in a surgical scrub?
3. Why are nosocomial infections often difficult to treat?
Protocols:
Hand Washing Exercise
Materials:

Organisms Media/Reagents Equipment

Normal Flora of the skin 1 TSA plates Marker
Soap
Alcohol Swabs

Methods:

1. Take 1 TSA plate.

2. Divide the plate in half.
3. Label each half as washed or unwashed.
4. Label each plate for identification and the exercise.
5. Using your knuckles, touch the plate on the unwashed half.
6. Either wash your hands or knuckles with soap, or swab with alcohol.
7. Repeat the touch on the plate on the washed half.
8. Incubate the plates at 370C overnight.
9. Observe growth.
 Disinfection Exercise

Materials:

Organisms Equipment Reagents/ Media

Staphylococcus aureus 12 culture tubes (13x100mm) Disinfectant:
Pseudomonas aeruginosa P1000 & P200 micropipettors Lysol
Pipette tips Bleach
Hockey Stick in EtOH Ethanol
Spinner Isopropanol
Vortex 12 TSA plates
TSB (10 mls)

Methods:

1. Label 6 culture tubes for Pseudomonas aeruginosa and 6 culture tubes for
Staphylococcus aureus.
2. Label 6 TSA plates for Pseudomonas aeruginosa and 6 TSA plates for Staphylococcus
aureus.
3. Choose a disinfectant. Add 1000 µl to each of 2 culture tubes labeled 20.
4. Working with a 500µl transfer volume and using TSB as the diluent, prepare 2 sets of
serial dilutions (1:2) of the disinfectant to 2-5. Remove and discard 500µl from the final
dilution. Each tube will contain 500µl.
5. Add 100 µl of S. aureus to each tube in the first set of dilutions.
6. Add 100 µl of P. aeruginosa to each tube in the second set of dilutions.
7. Incubate at 37°C for 10 minutes.
8. From each tube take 100µl of the dilution and transfer to the appropriately labeled TSA
plate.
9. Use the spread plate protocol on each plate, flaming the hockey stick between each
sample.
10. Incubate the plates at 37°C overnight.
11. Count colonies or describe the plate.
 Antibiotic Sensitivity

Materials:

Organisms Media/Reagents Equipment

Escherichia coli 4 TSA plates Bunsen burner
Staphylococcus aureus Antibiotics: Hockey Stick
Pseudomonas aeruginosa Tetracycline Spinner
Klebsiella pneumoniae Streptomycin Striker
Ampicillin Marker
Sterile water EtOH beaker
Filter Disks
Forceps
20µl pipettor + tips

Methods:

1. Take 4 TSA plates.

2. Divide each plate into four parts.
3. Label each part with one antibiotic or control (water).
4. Label each plate with one organism.
5. Use the spread plate protocol and spread each plate with 100µl of the appropriate
organism.
6. Place one filter disk in each quarter of each plate, flaming forceps before each placement.
7. Go over to antibiotic station.
8. Use a 20µl pipette to add 5µl of the appropriate antibiotic to each disk.
9. Incubate the plates at 37°C overnight.
10. Measure the zone of clearing.