1.  Bacterial Enumeration Gloria Phuong Le Microbiology Lab Dr. Fran Norflus

2. Introduction  Bacteria divide via binary fission but not all daughter cells are viable  Bacterial enumeration is the measurement of bacterial cells per unit of sample (volume or mass)  Counting of bacterial cells is important in:  Microbial ecology  Epidemiology

3. Methods  Total count vs. viable count: all cells vs. viable cells capable of producing progeny  There are 2 ways of counting bacterial cells: direct and indirect  Direct methods of enumeration involve counting actual cells or colonies  Indirect methods involve estimating the number of cells based on cell mass, scattering of light through a culture (spectroscopy), or through statistical method called most probable number (MPN)

4. Methods MethodEnumerationDescription Indirect and viableMPNStatistical estimation based on growth patterns Direct and viableStandard plate countSerial dilution of original sample and look for CFU Indirect and totalSpectroscopyMeasure the transmittance of sample Direct and totalStaining and microscopyStain the cells with fluorescent dyes to make them visible in raw samples

5. Microbes are everywhere  Foods can cause hazards to a person’s health if  They possess a large number of bacteria that can cause an infection by mere ingestion of the food  Contains microbial products of metabolism that can cause intoxication of the body  Food-handling techniques are important to prevent food poisoning  We will examine the presence of microbes in chopped beef and chicken samples  Normal and abnormal handling  Direct and viable method would be used

6. Procedures  Dilute the original sample (serial dilution)  Then use 0.1 ml of each dilution to perform the spread plate method (p. 56-59)  The plates will be incubated at 37 o C for 48 hours

7. Spread Plate Method

8. Direct and viable count  After 48 hours, there will be colonies growing on the plates  We will perform standard plate count (direct method to determine viable cells)  Each colony originated from one mother cell  We will count colony-forming-unit (CFU)  Record in your notes either  TNTC: too numerous too count  The exact CFU: 30-300

9. How to perform calculations?  First, we need to determine the concentrations of cells from countable plates  Then, we need to determine the dilution factor at each dilution (D f )  Next, we need to determine the total dilution (TD)  TD=D f1 xD f2 xD f3 …  Finally, to determine the concentration of cells in the original sample, we divide the number of CFU in the sample to the TD of that sample