Faculty of Medicine and Health Sciences Microbiology Lab Experiment 8 Bacterial Enumeration First Semester 2016-2017

Introduction: Bacterial enumeration is used for several purposes 1- Determine whether a substance or preparation complies with an established specification for microbiological quality, examples: Food microbiology (Milk, Juice, Ice-cream, meat, caned-food) Water microbiology 2- Determination of the number of bacterial cells on surfaces ( operation rooms) 3- Microbiological Experiments: preparation of bacterial suspensions with a particular concentration of bacterial cells: Antibiotic sensitivity Animal experiments ( when an animal is needed to be inoculated with a particular number of bacterial cells Note: The test is not applicable to products containing viable microorganisms as active ingredients…such as yogurt…

The enumeration of bacteria requires the use of small dilutions scientific notation that are routinely used in calculations. SCIENTIFIC NOTATION: In scientific notation, one digit (a number between 1 and 9) only is found to the left of the decimal point. The following examples are written in scientific notation: = 3.17 x 103 12,420,000 = 1.242 x 107 21,300 = 2.13 x 104 0.0047 = 4.7 x 10-3 0.000006 = 6.0 x 10-6

Estimation of Bacterial cells Concentration on Broth culture or Bacterial Suspension There are two methods can be used: 1- Direct counting of bacterial cells under the microscope 2- Plate method ( depend on colony counting) Examples: Bacterial grown in broth for an over night (or longer) Heavy bacterial suspensions ( prepared by suspending bacteria grown on plate in sterile water, sterile saline or sterile broth)

In broth cultures in in heavy bacteria suspensions, the number of bacterial cells can reach astronomical numbers/ml. So, if we take a drop of broth culture or bacterial suspension, place it on a slide and place a cover slip on it and then look under the microscope ( we may add Methylene blue to the bacterial suspension to stain the bacterial cells in order improve their visualization) under the microscope…it is not possible to count bacterial cells individually)

In case of taking a small volume of the bacterial growth culture of the heavy bacterial suspension, spread it on a plate and incubate for overnight (or longer time) In theory, each bacterial cell will give rise to a colony. But in this case, since we have a high number of bacterial cells cultured on the plate, ( CUTURE A SMALL VOLUME OF THE SAMPLE) the grown colonies will fuse together giving rise a lawn growth ( not individual colonies). So we can not count the colonies (plate method depends on counting the colonies)

So, whether direct bacterial cell ( count by under the microscope) or viable count (plate method/colony count) is going to be used the , the initial sample ( broth culture or bacterial suspension that contains high number of bacterial needs to be to be used so countable bacterial cells or colonies can be obtained .

Ten-Fold serial dilution (Used for bacterial cells enumeration): For Enumeration of Bacterial cells, 10 folds serial dilutions are needed to be prepared. Material needed: Bacterial broth culture or suspension 10 tubes each contains 9 ml sterile water or saline ( labeled 1 to 10) (containing Methylene blue) Automatic micropipettes Procedure: 1- One (1) ml of broth containing bacteria or 1 ml of prepared bacterial suspension is transferred and mixed with 9 ml of sterile water or saline in the first tube. The total volume in the first tube becomes 10ml ( a 1:10 dilution (also written 1/10 or 10-1, meaning 1/10 or 10-1 as many bacteria per ml as the original ml) or ( just the dilution factor of the tube number 1 is 10) ( 10 fold serial dilution)

2- Then, 1 ml of the first tube is transferred and mixed transferred to the second tube ( dilution factor is 102) ( 100 fold serial dilution) 3- Then, 1 ml of the second tube is transferred and mixed transferred to the third tube ( dilution factor is 1000 or 103)( 1000 fold) And so on……………………….until we reach the last tube ( dilution factor is 1010)  

Methods used for Bacterial Cells Enumeration: 1-Direct Microscopic Method (TOTAL CELL COUNT) In the direct microscopic count, a counting chamber (HEMOCYTOMETER) (or haemocytometer or counting chamber) (Petroff-Hausser counting chamber) is used The haemocytometer has a ruled area (or two areas) that are used to count the cells . Purpose of the haemocytometer: The haemocytometer (or haemocytometer or counting chamber) (Petroff-Hausser counting chamber) is a specimen slide which is used to determine the concentration of cells in a diluted liquid sample. It is frequently used to determine the concentration of blood cells (hence the name “hemo“) but also the concentration of sperm cells Or bacterial cells IN DILUTED SAMPLES .

Procedure: 1- Ensure both the haemocytometer and it's cover slip are clean. If they are dirty wash them in distilled water and then wipe them over with alcohol. When dry breath on the surface of the haemocytometer and quickly place the cover slip in position so that it is centered over the counting area of the chamber.

 2- Take (suck) up a small amount of the cell suspension ( say 7 fold dilution) to be counted in a fine tipped Pasteur pipette. Gently touch the pipette against the side of the cover slip where it touches the side of the cover slip. A drop of liquid should be drawn out of the pipette which will spread under the cover slip.

Each counting are of a 1 millimeter long (mm) by 1 mm wide and is 0 Each counting are of a 1 millimeter long (mm) by 1 mm wide and is 0.1 mm deep. Bacterial cells of the diluted sample are counted under the microscope in this area. The volume of the counted area would be: 1mm X 1mm X 0.1mm this will equal to 0.1 cubic mm or just simply a 0.1 micro litter So, the counted bacterial cells would be in 0.1 cubic mm or just simply a 0.1 micro litter

1- The counted of bacterial cells Say 100 cell in a volume of 0.1 cubic mm 2- To calculate the number of bacterial cell in 1 cubic mm: Multiply the counted of bacterial cells in 0.1 cubic mm (100 cell ) by 10 0.1 cubic mm contained 100 bacterial cells 1 cubic mm contains 100 bacterial cells X 10= 1000 3- To calculate the number of bacterial cell in cubic ml (1000 ul): Multiply the counted bacterial cell in 1 cubic mm by 1000 ( in point 2) 1000X 1000 = 1000000

1- To calculate the number of bacterial cells in the original sample Mutably the number of bacterial cell of point 3 by the dilution factor 107 1000000 X 107 = 1013 cell/ml The equation will be: Counted bacterial cell X 10 X 1000 X dilution factor Limitation of this procedure: 1- Can not distinguish whether the bacterial cells are dead or alive\ 2- It is difficult to perform in case the bacterium was of a kind that forms aggregates like staphylococci or streptococci

2-The Viable Count Method (The Plate Method): 1- A particular volume of a diluted bacterial suspension ( say 0.1 ml or just 100 ul) ( use the last five dilutions) is inoculated on a plate by using an automatic micropipette and then spread on nutrient agar plate using sterile glass spreader To sterilize the spreader, immerse it in 70% alcohol Light it up…and then allow to cool 9(be careful from the fire Use a new tip for each tube 2- Allow the plates to dry and place them in the incubator from overnight or longer 3- Count the colonies ( use two plates that show countable number of colonies (50-200)

3- Count the colonies ( use two plates that show countable number of colonies (50-200)

In theory: Each colony arise from a single or ( may be more) alive bacterial cells Count the colonies in at least two plates 1- Suppose that, you counted ( say colony), represent 70 bacterial cells that were plated in the previous day 2- The plated volume is 100 ul ( 0.1 ml) 3- The counted colonies will reflect the number of bacterial cells in 100 ul (0.1 ml) Say to got 70 colony. To calculate the number of bacterial cells in 1 ml (1000 ul), multiply the number of the counted colonies by 10 70 X 10 = 70 ( say this was of the diluted sample of a dilution factor 7) 4- To calculate the number of bacterial cells in the original sample, multiply the number of the counted colonies of the previous step by the dilution factor ( say this was of the diluted sample of a dilution factor 7) 70 X 10 7 = 7 X 10 8 colony forming unit (CFU)/ml ( since each colony may arise from more than one bacterial cell , CFU)/ml instead of cell/ml)

THE PLATE COUNT METHOD IS CALLED (VIABLE COUNT): why? Since it reflects the concentration of alive bacterial cells that give rise to colonies If you compare the concentration of bacterial cells of a bacterial, suspension counted by Direct microscopic counting Viable count Which method is most likely giver a higher number? Suppose you boiled the bacterial suspension before you starting the counting processes ( direct microscopic counting and viable count method). What would be your comments on the results? In other words, bacterial cell concentration obtained by direct microscopic and viable count method? What would be the result of the viable count method? explain

3- Turbidimetric Measurement: (Determination of bacterial cell concentration based on TURBIDITY) When bacteria grow in broth, the higher the bacterial cell number the more the broth becomes turbid If you prepare a bacterial suspension in sterile water, the more bacteria you add, the more the suspension will become turbid This is because a bacterial cells in liquids as a colloidal suspension that blocks and reflects light passing through the culture. You may place an appropriate volume of the bacterial broth culture or suspension in a transparent tube Within limits, using a spectrophotometer, the light absorbed by the bacterial suspension will be directly proportional to the concentration of cells in the culture.

By measuring the amount of light absorbed by a bacterial suspension (OD)) ( optical density (one can estimate and compare the number of bacteria present.) . The spectrophotometer: It consists of a light source, a filter which allows only a single wavelength of light to pass through, the sample tube containing the bacterial suspension, and a photocell that compares the amount of light coming through the tube with the total light entering the tube

The ability of the culture to block the light can be expressed as either percent of light transmitted through the tube or the amount of light absorbed in the tube The percent of light transmitted is inversely proportional to the bacterial concentration. (The greater the percent transmittance, the lower the number of bacteria.) The absorbance (or optical density) is directly proportional to the bacterial cell concentration. (The greater the absorbance, the greater the number of bacteria

Turbidimetric measurement is often correlated with some other method of cell count, such as the direct microscopic method or the plate count. In this way, turbidity can be used as an indirect measurement of the cell count. For example: 1. Several dilutions can be made of a bacterial broth culture 2. A Hemocytometer counter can then be used to perform a direct microscopic count on each dilution as mentioned earlier. 3. Then a spectrophotometer can be used to measure the absorbance of each dilution tube. 4. A standard curve comparing absorbance to the number of bacteria can be made by plotting absorbance versus the number of bacteria per ml 5. Once the standard curve is completed, any dilution tube of that organism can be placed in a spectrophotometer and its absorbance read. Once the absorbance is determined, the standard curve can be used to determine the corresponding number of bacteria per ml.

So, once you have the curve for a particular bacterium, next time when you have a broth culture of a suspension of this bacterium, just measure the OD using a spectrophotometer and then refer to the table to know the concentration of bacterial cells /ml McFarland standards are used as turbidity standards in the preparation of suspensions of microorganisms. The McFarland 0.5 standard has particular application in the preparation of bacterial inoculums for performing antimicrobial susceptibility testing.