LAB 2 EFFECT OF ANTIBIOTICS ON GROWTH OF Escherichia coli K12
Antibiotics are antimicrobial agents that 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 that 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 ( penicillin 7.5mg/ml), streptomycin (5 mg/ml) or chloramphenicol (10 mg/ml) or TSB
32 Lauria Broth Agar (LBA) 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 C A8000. 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 LB agar 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 plot to graph the plate count and linear plot 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 that 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.

Student #: _______________________________
CHEE 229 Lab 2 Questions (Hand in)
1. Which of these method(s) can be used to determine the number of viable microorganisms in a sample? a. light scattering in a spectrophotometer b. measuring colony forming units per ml c. measuring total cell mass d. counting a known volume of cells in a hemocytometer e. counting cell number with electronic counter
2. An agent that prevents the growth of bacteria without causing irreversible damage to the bacteria is referred to as _________________ but an agent that kills 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 wall synthesis is easily inhibited whereas animal cell wall synthesis is more resistant to the actions of the drugs. b. animal cells do not take up the drugs c. bacterial cell walls have a unique structure not found in eucaryotic host cells. d. animal cells inactivate the drugs before they can do any damage. e. all antibacterial agents kill all bacteria.
4. A growth medium that favors the growth of some microorganisms while inhibiting the growth of other microorganisms and contains substances which separates different bacteria because they show different growth characteristics with respect to these substances is a ___________________________ medium. a. selective b. differential c. selective and differential d. neither selective nor differential e. enriched and differential
5. 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.

6. (a) Which were the bactericidal agents in the current laboratory exercise?
(b)How did these bactericidal agents actually kill the bacteria? (ie., why did the bacteria die?)
7. Would these bactericidal agents be equally effective in killing bacteria if they are present in a biofilm? Explain.
8. Show a sample calculation for calculating colony forming units per ml.
9. Graph a set of results for all the antibiotics and the TB. Use a (a) semi-log plot for the plate count (i.e. log- or ln-scale on the y-axis and a linear scale for time on the xaxis) and (b) linear-linear plot for the absorbance results (i.e. linear scale on the x-axis and y-axis).