Determination of Phenol Coefficient of Disinfectants

There are many liquid chemicals that are used for cleaning of materials to lower the number of microorganisms. Such chemicals are called disinfectants e.g. lysol, phenol, hypochlorite (bleach), etc. However, the effectiveness of different disinfectants varies against a given microorganism. Therefore, phenol coefficient of these disinfectants by using a standard bacterial culture (e.g. Staphylococcus aureus) is determined. Hence, effectiveness of phenolics (phenol and its derivatives) is determined by comparing with phenol. The phenolic compounds kill bacteria by inactivating plasma membrane and enzymes, and denaturing the protein. The phenol coefficient is determined only of such chemicals which are bactericidal (but not bacteriostatic) in nature

Requirements

• Phenol dilution (1:10, 1:20, 1:30........1:90, 1:100) ,  
• Phenol derivative's dilution (1:100, 1:150, 1:200, 1:400, 1:450, 1:500) ,   
• Sterile nutrient broth tubes (20),
• Broth culture of Staphylococcus aureus, 
• Sterile pipette (1ml),   
• Inoculation loop, 
• Bunsen burner, 
• Test tube stand,

Procedure

1. Take the test tube stands each for phenol and phenolic compound to be tested.
2. Place test tubes of each concentration of each phenol and phenolic compound separately in the test tube stand.
3. Similarly, take equal number of sterile nutrient broth tubes (in other test tube stand) for each dilution of phenol and phenolic compounds. Label them according to dilution of disinfectant(s). Take one sterile nutrient broth tube as control.
4. After 5, 10 and 15 minutes of intervals, transfer aseptically one loopful bacterial culture from each nutrient broth tube appropriately labelled with the same dilution.
5. Similarly, also transfer one loopful fresh bacterial culture in control broth tube.
6. Incubate all the broth tubes inoculated with Staphylococcus aureus at 37°C for 48 hours. 

Phenol coefficient of the test chemicals = Reciprocal of the test chemical dilution marked/Reciprocal of the phenol dilution marked

                                  Suppose, the test chemical shows no growth at 10 minutes in 1:400 dilution, but growth at 5 minutes, and phenol shows no growth at 10 minutes in 1:80 dilution but no growth at 5 minutes, the phenol coefficient of test chemicals will be as below:

Phenol coefficient of test chemical= 1/400 divided by1/80 = 5,

Results

Observe the growth of Staphylococcus aureus in all the broth tubes treated with phenolic com- pounds and arrange the result as given in Table. Put mark on the highest dilution of phenol and the phenolic compounds tested that killed the bacteria in 10 minutes but not in 5 minutes and calcu- late the phenol coefficient as given below:

Disinfectant chemicals	DILUTION	Exposure time (min)
		5	10	15
Phenol	1:10-1:60	-	-	-
	1:70	-	-	-
	1:80	+	-	-
	1:90	+	+	-
	1:100	+	+	-
Phenolic derivative	1:100-1:350	-	-	-
	1:400	+	-	-
	1:450	+	+	-
	1:500	+	+	-

Table:- Effect of phenol and phenol derivatives on Staphylococcus aureus treated for different time intervals.

To determine the minimum inhibitory concentration (MIC) of Ampicillin against given bacterial sample.

Principle

Antibiotics are chemical compound produced by one type of microorganism which at low concentration selectively inhibit or kill another organism. The sensitivity of microbes to antibiotics can be expediently tested in the laboratory by lb broth diffusion method. In this method, the organism to be tested is heavily inoculated on the liquid medium in Test-Tubes. All Test-Tubes are filled with liquid media and varying concentration of antibiotics. The inoculated tubes are observed after incubation for a definite period of time forthe zone of inhibition. The diameters of zone of inhibition are measured. The inhibition is due to the diffusion of antibiotic from the media an area of high concentration to an area of low concentration. The smallest amount of the antibiotic needed to inhibit the growth of microorganism is called as MIC.

Requirements

• Lb broth media
• Sterile Test-tubes
• Ampicillin (antibiotics)
• Micropipette
• Bacteria culture

Procedure

1.       Prepare serial dilution of a ampicillin antibiotic as fellow:

12.5mg + 25ml DDW -- 0.5mg/ml............(a)

5ml (A)+ 5ml DDW -- 0.20mg/ml..........(b)

5ml (B)+ 5ml DDW -- 0.125mg/ml....... (c)

5ml (C)+ 5ml DDW -- 0.0625 .............. (d)

5ml (D)+ 5ml DDW -- 0.03125..............(e)

5ml (E)+ 5ml DDW -- 0.015625.............(f)

5ml (F)+ 5ml DDW -- 0.0078125............(g)

5ml (G)+ 5ml DDW -- 0.00390625.......... (h)

5ml (H)+ 5ml DDW -- 0.001953125.........(i)

2.       Arrange sterile tubes and no. Them (A) to (I)

3.       Take Equal amount of lb broth media in test tubes (3ml).

4.       Take 10ml bacterial sample and mixed it properly an all the test tubes (1ml in each test tube).

5.       Ampicillin solution.

6.       Incubate the tubes at 37ºC for 24 hours and observe it.

Observation

Zone of inhibition was observed around the well containing varying concentration of ampicillin.

S.No.	Test-Tube No.	(OD) after Incubate the tubes at 37ºC for 24 hours and observe it at 610nm,
1.	A	0.904
2.	B	0.559
3.	C	0.712
4.	D	0.748
5.	E	1.153
6.	F	1.219
7.	G	1.122
8.	H	0.985
9.	I	1.029

Fig: - : Determination of Minimum Inhibitory Concentration (MIC)

Result

The minimum inhibitory concentration expressed as the lowest concentration of ampicillin dilution for given bacterial culture.

Enumeration of bacteria (from soil): standard plate count

Background

Enumeration of bacteria is defined as the process of determining the number of bacteria in a given sample. Studies involving the analysis of materials, including food, water, milk, and—in some cases—air, require quantitative enumeration of microorganisms in the substances. Many methods have been devised to accomplish this, including direct microscopic counts, use of an electronic cell counter such as the Coulter Counter, chemical methods for estimating cell mass or cellular constituents, turbidimetric measurements for increases in cell mass, and the serial dilution–agar plate method. Serial dilution–agar plate method will be adopted here

Principle

Microorganisms are ubiquitous and can be present in thousands or millions in a sample, making it difficult to count their numbers. However, serially diluting the cultures makes it easier to determine the count. This method involves serial dilution of a bacterial suspension in sterile water blanks, which serve as a diluent of known volume. After serial dilution, the aliquots of the diluted sample are plated on an appropriate culture media. Then, the plates are incubated, after which the number of colonies formed is counted. This technique is also known as plate count or colony counts. This method is based on the fact that, on a solid nutrient medium, the viable cells (a cell which is able to divide and form a colony) grow to form colonies that are visible to the naked eye. The resultant colonies are visible to the naked eye making visualization, selection and counting simpler. Plates suitable for counting must contain between 30-300 colonies. To arrive at a suitable dilution that results in this number, serial dilution method is used. The method involves making a tenfold dilution at a time by taking a small amount of the original sample (For e.g. 1 mL) and making up the volume (to say 10 mL) by adding an appropriate buffer or normal saline. For every dilution, a specific volume is plated on a suitable medium.                                                
Plate count method can be of two types:

Pour plate method: Molten agar, cooled to 45°C, is poured into a Petri dish containing a specified amount of the diluted sample. Following addition of the molten-then-cooled agar, the cover is replaced, and the plate is gently rotated in a circular motion to achieve uniform distribution of microorganisms. This procedure is repeated for all dilutions to be plated. Dilutions should be plated in duplicate for greater accuracy, incubated overnight, and counted on a Quebec colony counter either by hand or by an electronically modified version of this instrument. This technique allows the growth of anaerobes (beneath the surface) as well as aerobes (on the surface of the plate).

Spread plate method: A known volume of the dilution is plated on the nutrient agar plate. The colonies obtained can be selected and sub cultured easily for further use. The bacterial count in the original sample is estimated as CFU/mL (Colony Forming units/mL).                                                     
CFU/mL = CFU/volume of dilution plated × Dilution factor

Materials required

Sample (soil), dehydrated nutrient agar powder, Erlenmeyer flasks, culture tubes, Petri plates, spreader, beakers, 90% ethanol, 0.8% NaCl, micropipettes, test tube stand, autoclave, laminar air flow, bacteriological incubator, water bath set at 50°C.

Methodology Preparations

Prepare 400ml of nutrient agar (in Erlenmeyer flasks), 5 numbers of 9ml saline blanks, a 99ml saline blank (in Erlenmeyer flask), 20 Petri plates and autoclave.

Sample collection

Collect ~2g of garden soil sample in a sterile polyethylene bag using ethanol sterilized spatula. Weigh 1g of soil and keep them inside laminar flow, until further use.

Serial dilution and pour plate

1.  Following autoclaving, cool the molten agar in a water bath maintained at 50°C.                                        
2.  Transfer the sterilized Petri plates, saline, serial dilution tubes and micropipettes to ready-to-work (surface sterilized (using ethanol) and UV sterilized for 15 minutes) laminar air flow.                                        
3.   Cool the sterile 99ml 0.8% saline blank, and add 1g of soil and mix well. This dilution is regarded as 100 times dilution (10-2).
4.  Label the saline blank in flask as dilution 2 and five 9-ml saline blank tubes as dilution 3 through 7.                              
5.  Place the labelled tubes in a test tube rack. Label the Petri dishes in duplicates as P3, P4, P5, P6, P7 (pour plates) and S3, S4, S5, S6, S7 (spread plates).
6. With a sterile pipette, aseptically transfer 1 ml from the 99ml saline blank mixed with soil sample to tube 3. The sample has been diluted 1000 times to 10-3. Discard the tip.  
7.  Using sterile tip, add 0.1ml each from 3rd tube to 2 empty P1 plates and leave it aside
8.  Mix the mixture in the 3rd tube by gently pipetting up and down. Using a fresh pipette tip, transfer 1ml from tube 3 to tube 4. Now the sample has been diluted 10,000 times to 10-4. Discard the tip.
9. Using sterile tip, add 0.1ml each from 4th tube to 2 empty P2 plates and leave it aside.        
10.  Using a fresh sterile tip, mix the mixture in the 4th tube by gently pipetting up and down. Using a fresh pipette tip, transfer 1ml from tube 4 to the tube 5. Now the sample has been diluted 100,000 times to 10-5.
11.  Using sterile tip, add 0.1ml each from 5th tube to 2 empty P3 plates and leave it aside.                         
12.  Using a fresh sterile tip, mix the tube 5 by gently pipetting up and down. Using a fresh pipette tip, transfer 1ml from tube 5 to the tube 6. Now the sample has been diluted 10, 00,000 times to 10- 6.
13.  Using sterile tip, add 0.1ml each from 6th tube to 2 empty P4 plates and leave it aside.  
14.  Using a fresh sterile tip, mix the tube 6 by gently pipetting up and down. Using a fresh pipette tip, transfer 1ml from tube 6 to the tube 7. The sample has been diluted 100, 00,000 times to 10-7. Dilution process is now complete.                                                                   
15.  Using sterile tip, add 0.1ml each from 7th tube to 2 empty P5 plates and leave it aside.    
16.   Check the temperature of the molten agar medium to be sure the temperature is above 45°C. Remove a media from the water bath and wipe the outside surface dry with a paper towel.
17.    Using the pour-plate technique, pour the agar into Plate P1 through P5 1A and rotate the plate gently to ensure uniform distribution of the cells in the medium.       
18.    Once the agar has solidified, incubate the plates in an inverted position for 24 hours at 37°C.
19.   Add and spread 0.1 ml of sterile saline in a nutrient agar plate to act as negative control. Open and leave a nutrient agar plate at the corner of the laminar to act as laminar control. Incubate theses controls along with other plates.

Spread plate technique

The spread-plate technique requires that a previously diluted mixture of microorganisms be used. During inoculation, the cells are spread over the surface of a solid agar medium with a sterile, L-shaped bent glass rod while the Petri dish is hold on hand or spun on a lazy Susan/turntable. The step-by-step procedure for this technique is as follows:

1.  Prepare soil sample suspensions as described above and label agar plates accordingly.
2.  Place the bent glass rod into a beaker and add a sufficient amount of 95% ethyl alcohol to cover the lower, bent portion.
3.  Place an appropriately labelled nutrient agar plate on the turntable. With a sterile pipette, add 0.1 ml of soil suspension on the center of the plate.
4.  Remove the glass rod from the beaker, and pass it through the Bunsen burner flame with the bent portion of the rod pointing downward to prevent the burning alcohol from running down your arm.
5.  Allow the alcohol to burn off the rod completely. Cool the rod for 10 to 15 seconds.
6.  Open the Petri plate lid and touch the agar surface with sterilized L-rod. Move the rod up, down and in side ways to spread the sample.
7.  Turn the plate at 90° angle and repeat the spreading process. Again turn the plate at 90° angle and do the spreading. For the last time, turn again the plate at 90° angle and do the spreading.
8.   Immerse the rod in alcohol and re-flame.
9.  Close the plate and allow the sample to adsorb on the agar surface for 3 minutes. Incubate the plates in an inverted position for 24 hours at 37°C.
10.  Add and spread 0.1 ml of sterile saline in a nutrient agar plate to act as negative control and open and leave a nutrient agar plate at the corner of the laminar to act as laminar control. Incubate theses controls along with other plates.

Result

Following 24hours incubation, sample and laminar control remained sterile. Pour and spread plates contained approximately 106 CFU/g and 107 CFU/g of soil, respectively. 

Antimicrobial sensitivity test

Background

Chemotherapeutic agents are chemical substances used to treat infectious diseases. Their mode of action is to interfere with microbial metabolism, thereby producing a bacteriostatic or bactericidal effect on the microorganisms, without producing a like effect in host cells. Chemotherapeutic agents act on a number of cellular targets. Their mechanisms of action include inhibition of cell-wall synthesis, inhibition of protein synthesis, inhibition of nucleic acid synthesis, disruption of the cell membrane, and inhibition of folic acid synthesis. These drugs can be separated into two categories:   

1. Antibiotics are synthesized and secreted by some true bacteria, actinomycetes, and fungi that destroy or inhibit the growth of other microorganisms. Today, some antibiotics are laboratory synthesized or modified; however, their origins are living cells.                                                      
2. Synthetic drugs are synthesized in the laboratory

Principle

The available chemotherapeutic agents vary in their scope of antimicrobial activity. Some have a limited spectrum of activity, effective against only one group of microorganisms. Others exhibit broad-spectrum activity against a range of microorganisms. The drug susceptibilities of many pathogenic microorganisms are known, but it is sometimes necessary to test several agents to determine the drug of choice. A standardized diffusion procedure with filter paper discs on agar, known as the Kirby-Bauer method, is frequently used to determine the drug susceptibility of microorganisms isolated from infectious processes. This method allows the rapid determination of the efficacy of a drug by measuring the diameter of the zone of inhibition those results from diffusion of the agent into the medium surrounding the disc. In this procedure, filter-paper discs of uniform size are impregnated with specified concentrations of different antibiotics and then placed on the surface of an agar plate that has been seeded with the organism to be tested.

Materials required

Test culture: 0.85% saline suspensions adjusted to an absorbance of 0.1 at 600 nanometer (nm) or equilibrated to a 0.5 McFarland Standard, Mueller-Hinton agar plates, antibiotic discs, forceps, Bunsen burner, sterile cotton swaps, 70% ethanol, millimetre ruler.

Protocol

1.  Place MHA plates right-side-up in an incubator heated to 37°C for 10 to 20 minutes, allowing the plates to warm up.
2. Label the bottom of each of the agar plates with the name of the test organism or strain to be inoculated.
3.  Using aseptic technique, inoculate all agar plates with their respective test organisms as
4.  Follows; Dip a sterile cotton swab into a well-mixed saline test culture and remove excess inoculum by pressing the saturated swab against the inner wall of the culture tube.
5.  Using the swab, streak the entire agar surface horizontally, vertically, and around the outer edge of the plate to ensure a heavy growth over the entire surface.
6.   Allow all culture plates to dry for about 5 minutes.
7.  Distribute the individual antibiotic discs (maximum of 5 discs in 15mm plates) at equal distances (~2.5 cm) with forceps dipped in alcohol and flamed.
8.  Gently press each disc down with the wooden end of a cotton swab or with sterile forceps to ensure that the discs adhere to the surface of the agar. Note: Do not press the discs into the agar.
9.   Incubate all plate cultures in an inverted position for 24 to 48 hours at 37°C.
10.   Following incubation, examine all plate cultures for the presence or absence of a zone of inhibition surrounding each disc.
11.  Using a ruler graduated in millimeters, carefully measure each zone of inhibition to the nearest millimeter. Tabulate the results.
12.   Compare your results with Table and determine the susceptibility of each test organism to the chemotherapeutic agent.

	Measurements in millimeter
Antibiotic disc	Ampicillin (A) 10µg	Erythromycin (E) 10µg
Test culture	R	S(16mm)

S = sensitive; R = resistant; I = intermediate

Result

The test culture was resistant to ampicillin, clindamycin, nystatin, intermediate to amoxicillin and sensitive to streptomycin.