LAB 2 EFFECT OF ANTIBIOTICS ON GROWTH OF Escherichia coli K12
Antibiotics are antimicrobial agents which either kill bacteria outright (bactericidal)
or inhibit growth without killing (bacteriostatic). A given antibiotic is either bactericidal or
bacteriostatic but not both. They can be naturally-occurring products which are
produced by other microorganisms, semisynthetic agents which are chemically modified
versions of naturally-occurring products, or wholly synthesized in the Lab In any case,
they have defined targets within the bacterial cell and specific, though at times poorly
understood, mechanisms of action. β-lactam antibiotics such as the penicillins (including
ampicillin) block peptidoglycan biosynthesis while the aminoglycoside antibiotics (e.g.
streptomycin) and chloramphenicol target the ribosome and inhibit protein synthesis. In
the current exercise, the influence of these three antibiotics on the growth of E. coli K12
will be assessed using both spectrophotometric and plate count methods of bacterial
cell enumeration.
Spectrophotometric determination of bacterial numbers in aqueous suspension is
determined using optical density measurements. A beam of light is passed through a
cell suspension and the amount of light transmitted by/through the suspension is
measured. The amount of light transmitted is inversely proportional to the number of
organisms present, though only within a defined range of cell concentration.
Plate count methods of determining bacterial cell numbers rely on bacterial
viability at the time of cell enumeration and are, thus, also referred to as viable counts.
Viable or plate counts are based on the assumption that each bacterium in a
suspension will give rise to a single colony when the suspension is diluted appropriately
and plated onto solid growth medium. Not all colonies arise from a single cell, however,
since certain microorganisms tend to clump or aggregate in suspension. Thus, when
plated onto solid medium these clumps or aggregates will give rise to a single colony
and actual viable cell numbers will be underestimated. For this reason, the results
obtained from viable cell counts are usually expressed as colony forming units (CFU)/ml
rather than cells/ml.
Materials: (per group of 4)
1 tube of log phase Escherichia coli CA8000
1 pinocchio flask with 50 ml TSB
1 tube of either an antibiotic (ampicillin 2.5 mg/ml), streptomycin (5 mg/ml) or
chloramphenicol (10 mg/ml) or TSB
32 TSBA plates
40 tubes with 0.9 ml sterile water
32 tubes of sterile glass beads
Procedure:
1.
Select a flask, label and place in the shaker bath to warm up. Allow 5 minutes for
the flask to equilibrate to temperature and then add 2 ml of E. coli CA8000. This
is time 0. Return the flask to the shaker bath.
2.
Set up a rack of tubes of 0.9 ml sterile water so that one can perform 10 -1 to 10-5
dilutions at each time point. Do not write on the caps. Write on the glass only.
Label plates with name, time, antibiotic and dilution so that all are ready to use at
the appropriate time.
3.
At time 20 minutes and at 20 minute intervals thereafter, read the absorbance at
a wavelength of 550 nm, by inserting the side arm of the flask into the
spectrophotometer. Use a flask of TSB for a blank. (It is very important to keep
the cells at 37°C so they will continue to grow and so the flask should be out of
the waterbath or air bath in as short of a time as possible).
4.
As well at times, 20, and at 20 minute intervals thereafter, a 0.1 ml sample
should be removed and added to 0.9 ml of sterile water.
5.
This sample should be diluted serially up to 10-5. After the dilutions are
completed, 0.1 ml samples of 10-3, 10-4, 10-5 dilution should be spread on TSBA
plates using sterile beads.
6.
At time 80 minutes add 1.0 ml of either the appropriate antibiotic solution or TSB
immediately after taking the reading and sample. (The antibiotic to be studied will
be assigned to each group by the demonstrator.)
7.
Continue to take sample readings at 20 minute intervals as above, up to 140
minutes if possible. However, ALL DILUTIONS need to be plated after the
addition of the antibiotic. Groups that added TSB continue to plate only 10-3 to
10-6 dilutions.
8.
Incubate plates overnight at 37°C. Count colonies and post results as colony
forming units per ml (CFU/ml) or give to your demonstrator as instructed.
9.
Graph a set of results for all the antibiotics and the TSB. Use semi-log paper to
graph the plate count and linear paper for the absorbance results.
Student #: _______________________________
Growth Lab Questions (Hand in)
1.
An agent that kills bacteria is referred to as __________.
2.
An agent that prevents the growth of bacteria without causing irreversible
damage to the bacteria is referred to as __________.
3.
The most selective antibacterial agents are those that interfere with bacterial cell
wall synthesis. This is because
a.
bacterial cell walls have a unique structure not found in eucaryotic host
cells.
b.
bacterial cell wall synthesis is easily inhibited whereas animal cell wall
synthesis is more resistant to the actions of the drugs.
c.
animal cells do not take up the drugs.
d.
animal cells inactivate the drugs before they can do any damage.
4.
A growth medium that favors the growth of some microorganisms but inhibits the
growth of other microorganisms is a __________ medium.
a.
selective
b.
differential
c.
selective and differential
d.
neither selective nor differential
5.
Which of the following antibiotics specifically inhibits DNA synthesis?
a.
gentamycin
b.
polymyxin B
c.
quinolones
d.
tetracycline
6. True or False
One way in which organisms may exhibit resistance to a drug is by
pumping the drug out of the cell immediately after it has entered.
7.
The exponential growth rate of a bacterial culture is affected by all of the
following except
a.
the genetic potential of the organism.
b.
the composition of the growth medium.
c.
the number of cells initially inoculated into the culture vessel.
d.
the condition of incubation.
8. a) How did the bactericidal agents in the current laboratory exercise actually kill the
bacteria? (ie., why did the bacteria die?)
b) List two additional bactericidal antibiotics.
9.
Show a sample calculation for calculating colony forming units per ml.
10.
Graph a set of results for all the antibiotics and the TB. Use semi-log paper to
graph the plate count and linear paper for the absorbance results.
DEMONSTRATION 2
Various types of media are used as a means of preliminary identification of specific
bacteria.
Some of these media are as follows:
1. Selective
Selective media are those media which contain specific chemical substances that
inhibit certain bacteria while allowing others to grow. For example, Staphylococcus
aureus will grow on agar containing 7.5% NaCl while E. coli will not.
Observe the two salt agar plates inoculated with these organisms. Salt agar is
selective for Staphylococci. Compare the amount of growth of S. aureus on salt agar
to that on nutrient type agar. In the selectivity luxuriance of growth may be sacrificed.
2. Differential
A differential medium contains substances which aid in the separation of different
types of bacteria because certain bacteria show different growth characteristics with
respect to these substances. For example, MacConkey agar is a differential plating
medium for the selection and recovery of Enterobacteriaceae and related enteric
Gram negative rods. Bile salts and crystal violet are included to inhibit the growth of
Gram positive bacteria and some fastidious Gram negative bacteria. Lactose is the
sole carbohydrate. Lactose fermenting bacteria produce colonies that are varying
shades of red because of the conversion of the neutral red indicator dye (red below
pH 6.8) from the production of mixed acids. Colonies of nonlactose fermenting
bacteria appear colourless or transparent.
3. Enriched
The addition of components like blood, serum, amino acids, vitamins, proteins etc.
provides extra nutrients so that the medium is referred to as enriched. Note the
growth of Streptococcus pyogenes on tryptic soy agar and then on blood agar, an
enriched medium.
4. Synthetic
The ingredients of synthetic media are known i.e. such a medium is chemically
defined. Such media are widely used in studying metabolic patterns and genetics of
microorganisms. They may be used in assays and antibiotic tests. Synthetic media
often may give less luxuriant growth of a particular organism than a less well defined
medium. Compare growth of the demonstration species on a synthetic and nutrient
medium.