Question

1. Antibiotic Evaluation

The following set of data was taken from tests of antibiotics on two organisms isolated from the lungs of a patient with Cystic Fibrosis. These evaluations were carried out using the disk diffusion method, and the diameter of the zone of inhibition (ZOI) is provided for each organism and antibiotic.

Pseudomonas aeruginosa is Gram-negative.

Corynebacterium xerosis is Gram-positive.

Resistant (R): ZOI ≤ 11 millimeters (mm)
Intermediate (I): ZOI > 11 mm and ≤ 17 mm
Sensitive (S): ZOI > 17 mm

Instructions: Evaluate the susceptibility of each organism to the antibiotics listed. Then, fill in the “Evaluation” columns with R, I, or S based on your analysis.

Antibiotic	Pseudomonas aeruginosa		Corynebacterium xerosis
	ZOI, mm	Evaluation	ZOI, mm	Evaluation
Penicillin	3		1
Erythromycin	12		33
Rifampin	11		2
Ciprofloxacin	19		27

Based on the results from these two organisms and the data provided, determine which antibiotics are broad-spectrum and which antibiotics are narrow-spectrum.

Antibiotic	Spectrum
Penicillin
Erythromycin
Rifampin
Ciprofloxacin

2. Media preparation

2A. Of all the infectious agents we have discussed, at least one of them can remain pathogenic after going through a correctly-functioning autoclave. Which is it?

2B. To test our autoclaves at Southwestern we perform a monthly “Endospore viability” run. Explain why this is a good way to determine if the autoclave is working properly.

3. Estimation of Bacterial Population Size

3A. Estimate the population of the original culture based on the information below:

                Number of colonies on plate: 74
                Volume plated: 0.1 mL
                Dilution of culture plated: 1/1,000,000 (1 x 10^-6)

Estimated population density (Be sure to include the correct units!):

3B. If I took 0.5 mL of the original culture and diluted it into 15 mL of water, how much would the original culture be diluted?

3C. Suppose that I took 1 mL of water from Sweetwater Reservoir and plated it onto TSA. After 1 day of growth, I counted 30 colonies on the plate. Sweetwater Reservoir is estimated to have approximately

                34,000,000,000,000 milliliters of water (3.4 x 10¹³ mL)

How many bacteria do you estimate are living in Sweetwater Reservoir?

Answer 1

1. Given the ZOI range to evaluate the effect of antibiotic on Pseudomonas aeruginosa and Cornebacterium xerosis.[Resistant (R) </= 11 mm; Intermediate (I) > 11 mm and </= 17 mm; Sensitive (S) > 17 mm]

Antibiotic	Pseudomonas aeruginosa	Cornebacterium xerosis
ZOI, mm	Evaluation	ZOI, mm	Evaluation
Penicillin	3	R	1	R
Erythromycin	12	I	33	S
Rifampicin	11	R	2	R
Ciprofloxacin	19	S	27	S

Both the bacteria are resistant to Penicillin and Rifampicin as the ZOI is </= 11 mm; P. aeruginosa is intermediate with erythromycin as the ZOI is below 17 mm and > 11 mm whereas C. xerosis is sensitive as the ZOI is way above 17 mm. Both the bacteria are sensitive to Ciprofloxacin as the ZOI are > 17 mm.

Antibiotic	Spectrum
Penicillin	Narrow
Erythromycin	Broad
Rifampicin	Narrow
Ciprofloxacin	Broad

Broad spectrum antibiotics ar effective over a broad range of bacteria whereas narrow spectrum antibiotics are effective over a narrow range of bacteria. Here, P. aeruginosa is a gram negative bacteria whereas C. xerosis is a gram positive bacteria. The antibiotic that is effective against both the types of bacteria can be considered broad spectrum, and those that is ineffective to one or both of them can be considered as narrow spectrum.

Penicillin and Rifampicin are ineffective against both the bacteria and therefore can be considered narrow spectrum. Erythromycin and Ciprofloxacin are effective against both the bacteria and therefore can be considered broad spectrum.

2A. Autoclaving results in the hydrolysis and coagulation of proteins in microbial cells. According to Markova et al., (2010), however, certain bacteria such as E. coli tend to survive by existing in what is known as a polymorphic L- form, where they lose the ability to form cell walls and propagate without them and tide over unfavorable conditions.

2B. Endospores can survive for several hours at 100 degrees C. The temperature used in an autoclave is 120 degrees C which should kill endospores. If the autoclave does not function properly, if it is not able to maintain pressure as a result of which the temperature is not maintained, then it would show in the endospore viability assay results.

3A. we are asked to estimate the bacterial population in the original culture.

Given the number of colonies on the plate = 74

Volume plated = 0.1 mL

Diltution of the cutture = 10^-6. This means the culture is diluted 10⁶ times. This is the Dilution Factor or DF, which is the inverse of the diltuion.

We would determine the population using the formula for CFU (Colony Forming Units):

CFU / mL = No. of colonies * DF / volume plated

Incorporating the values given in the above formula, we get:

CFU / mL = 74 * 10⁶ / 0.1 = 740 * 10⁶ = 7.4 * 10⁸.

Therefore, the population in the original culture is 7.4 * 10⁸ CFU / mL.

3B. The original culture, 0.5 mL is taken and diluted to a final volume of 15 mL.

the dilution here would be: 0.5 mL / 15 mL = 5 mL / 150 mL = 1 mL / 30 mL.

Therefore, the dilution used here is 1 / 30.

In other words, the original culture is diluted 30 times (DF = 30).

3C. Given that 1 mL of the water sample gave 30 colonies. And the total volume of water in the reservoir is 3.4 x 10¹³ mL.

In this case, the DF is 1, as the sample is plated directly. Also, the volume plated is 1 mL. So we have to simply multiply the number of colonies with the total volume of the reservoir.

CFU / mL = 30 * 1 / 1 = 30 CFU / mL

Total number of bacteria in the reservoir = 30 * 3.4 x 10¹³  = 102 x 10¹³  = 1.02 *  x 10¹⁵ CFU.

The number of bacteria in the entire reservoir is 1.02 *  x 10¹⁵ CFU.