Presentation on theme: "Microbial Count Aim: Count the number of bacterial cells in a provided sample Methods: Total count, Viable count I. Total count This technique involves."— Presentation transcript:
1 Microbial CountAim: Count the number of bacterial cells in a provided sampleMethods: Total count, Viable countI. Total countThis technique involves the count of all bacteria in agiven suspension either viable or dead. This can beperformed either:1) TurbidimetryTotal number of bacteria is calculated from a standard calibration curve which is obtained by plotting log bacterial count of a suspension against absorbance of this suspension.
2 microbial cells is the direct counting of cell suspension in 2) Counting chamberThe most common method of enumerating the totalmicrobial cells is the direct counting of cell suspension ina counting chamber of known volume using amicroscope. There are many types of counting chambersfrom which the most common type is calledhemocytometer (originally designed for performingblood cell counts). It consists of a thick glass microscopicslide with a rectangular groove that creates a chamberof known dimensions.
3 This chamber is marked with a counting grid (network) of perpendicular lines divided into:- Nine large squares (1mm x 1mm)- The center square is divided into 25 smaller squares(0.2mm x 0.2mm).- Depth of the counting chamber : 0.1 mm.Therefore it is possible to count the number of cells ina specific volume of fluid (10-20 ul in each slide notch),and thereby calculate the concentration of cells in theoriginal fluid volume.
8 II. Viable count Pour plate (seed) method Principle High numbers of bacteria are often present in samples.Some samples may contain up to 1010 bacteria /ml. Atthis concentration, a 0.1 ml volume plated onto anutrient agar plate could potentially yield one billioncolonies which is impossible to count in a Petri dish.To overcome this problem, serial dilutions of the sampleprior to plating reduce the number of organisms suchthat when a small volume of the diluted sample is plated,the colonies are countable. For statistical accuracy, onlyplates containing between 30 and 300 colonies arecounted.
9 Procedure:1. Make a serial dilution of the supplied bacterial culture by transferring aseptically exactly 1 ml amounts (using a sterile 1 ml pipette) into the sterile test tubes containing 9 ml saline solutions.2. Each of the tubes must be thoroughly mixed (by means of a new 1 ml pipette) to insure even distribution of the organism.3. With a sterile 1 ml pipette, aseptically transfer 1 ml of each dilution into each of 3 sterile Petri dishes. Mark each plate with the dilution present.4. Melt the nutrient agar, pour into Petri dishes, and mix the bacterial suspension with the nutrient agar.5. Incubate; count-the number of colonies choosing plates that have between 30 and 300 colonies.
11 Precautions for a succesful experiment: 1) Good mixing of the m.o. both in:a) the 9-ml saline tubesb) the Petri dishes together with melted nutrient agar.2) Melted agar tubes must be poured at a temp. not too high (to avoid killing of the m.o.) & not too low (to allow for a sufficient mixing time that will insure an even distribution of the m.o. throughout the plate).3) Petri dishes must be inverted upside-down immediately after solidification of poured nutrient agar to prevent condensed water vapor droplets in the inner side of the plate lid from falling-down over the agar surface, otherwise, wet agar surface will cause overlapping of the microbial colonies appearing as an uncountable patch over agar surface.
13 2) Membrane filtration method In this method cells are filtered onto membrane filter which is then placed over nutrient agar surface plates. Number of c.f.u/ml is determined after incubation at suitable temp. & time.