M ICROBIAL G ROWTH AND E NUMERATION

O BTAINING P URE C ULTURES  Pure Culture: Contains a single microbial species.  Most clinical and environmental specimens contain several different microorganisms.  To obtain a pure culture, individual organisms must be isolated.  The most common method of isolation is the streak plate, in which a sterile loop is inserted into a sample and streaked onto a plate in a pattern, to obtain individual colonies  A colony is a population of cells arising from a single cell or spore or from a group of attached cells.

T HE S TREAK P LATE M ETHOD

G ROWTH OF B ACTERIAL C ULTURES  Bacterial Division: Occurs mainly by binary fission.  A few bacterial species reproduce by budding.  Generation Time: Time required for a cell to divide and its population to double.  Generation time varies considerably among organisms & with environmental conditions, such as temperature. E. coli divides every 20 minutes. Most bacteria divide every 1 to 3 hours. Some bacteria require over 24 hours to divide.

B ACTERIAL G ROWTH : B INARY F ISSION

G ROWTH OF B ACTERIAL C ULTURES Logarithmic Representation of Bacterial Growth : We can express the number of cells in a bacterial generation as 2 n, where n is the number of doublings that have occurred.

C ELL D IVISION

B ACTERIAL G ROWTH C URVE

T HE MATHEMATICS OF EXPONENTIAL GROWTH

P HASES OF G ROWTH Bacterial Growth Curve : When bacteria are inoculated into a liquid growth medium, we can plot of the number of cells in the population over time. Four phases of Bacterial Growth: Lag Phase: For a while, the number of cells changes very little because the cells do not immediately reproduce in a new medium. Period of adjustment to new conditions. Little or no cell division occurs, population size doesn’t increase. Phase of intense metabolic activity, in which individual organisms grow in size. May last from one hour to several days.

P HASES OF G ROWTH Log Phase: Cells begin to divide. Period of most rapid growth. Number of cells produced > Number of cells dying Cells are at highest metabolic activity. Cells are most susceptible to adverse environmental factors at this stage. Radiation Antibiotics

P HASES OF G ROWTH Stationary Phase: Population size begins to stabilize. Number of cells produced = Number of cells dying Overall cell number does not increase. Cell division begins to slow down. Factors that slow down microbial growth: -Accumulation of toxic waste materials -Acidic pH of media -Limited nutrients -Insufficient oxygen supply

P HASES OF G ROWTH Death or Decline Phase: Population size begins to decrease. Number of cells dying > Number of cell produced Cell number decreases at a logarithmic rate. Cells lose their ability to divide. A few cells may remain alive for a long period of time.

F OUR P HASES OF B ACTERIAL G ROWTH C URVE

D IRECT M EASURMENT OF M ICROBIAL G ROWTH

P LATE C OUNT  Most frequently used method of measuring bacterial populations.  A plate count is done by either the pour plate method or the spread plate method. Assumptions: Each colony originates from a single bacterial cell. Original inoculum is homogeneous. No cell aggregates are present. Advantages: Measures viable cells. Disadvantages: Takes 24 hours or more for visible colonies to appear. Only counts between 25 and 250 colonies are accurate. Must perform serial dilutions to get appropriate numbers/plate.

S ERIAL D ILUTIONS ARE U SED WITH THE P LATE C OUNT M ETHOD TO M EASURE N UMBERS OF B ACTERIA

P LATE C OUNT A. Pour Plate: Either 1.0 ml or 0.1 ml of dilutions of the bacterial suspension is introduced into a Petri dish. The nutrient medium, in which the agar is kept liquid by holding it in a water bath at about 50°C, is poured over the sample, which is then mixed into the medium by gentle agitation of the plate. When the agar solidifies, the plate is incubated. With the pour plate technique, colonies will grow within the nutrient agar (from cells suspended in the nutrient medium as the agar solidifies) as well as on the surface of the agar plate. Disadvantages: Not useful for heat sensitive organisms. Colonies appear under agar surface.

P LATE C OUNT B. Spread Plate A 0.1 ml inoculum is added to the surface of a prepoured, solidified agar medium. The inoculum is then spread uniformly over the surface of the medium with a specially shaped, sterilized glass or metal rod.

P OUR P LATES VERSUS S PREAD P LATES

F ILTRATION -Used to measure small quantities of bacteria. Example: Fecal bacteria in a lake or in ocean water. -A large sample (100 ml or more) is filtered to retain bacteria. -Filter is transferred onto a Petri dish. -After incubation colonies are counted.

F ILTRATION

C OUNTING B ACTERIA BY M EMBRANE F ILTRATION

M OST P ROBABLE N UMBER (MPN)  Used mainly to measure bacteria that will not grow on solid medium.  A sample is diluted repeatedly and several broth tubes are inoculated for each dilution point.  The number of positive tubes in each set is counted.  Statistical method: Determines 95% probability that a bacterial population falls within a certain range.

M OST P ROBABLE N UMBER (MPN)

D IRECT M ICROSCOPIC C OUNT In the method, a measured volume of a bacterial suspension is placed within a defined area on a microscope slide. A 0.01 ml sample is spread over a marked square centimeter of slide, stain is added so that the bacteria can be seen, and the sample is viewed under the oil immersion objective lens. The area of the viewing field of this objective can be determined. Once the number of bacteria has been counted in several different fields, the average number of bacteria per viewing field can be calculated. From these data, the number of bacteria in the square centimeter over which the sample was spread can also be calculated. Because this area on the slide contained 0.01 ml of sample, the number of bacteria in each milliliter of the suspension is the number of bacteria in the sample times 100. A specially designed slide called a Petroff-Hausser cell counter is also used in direct microscopic counts

D IRECT M ICROSCOPIC C OUNT Advantages: No incubation time required. Disadvantages: Cannot always distinguish between live and dead bacteria. Motile bacteria are difficult to count. Requires a high concentration of bacteria (10 million/ml).

D IRECT M ICROSCOPIC C OUNT

I NDIRECT M ETHODS OF M EASUREMENT

T URBIDITY As bacteria multiply in media, it becomes turbid. In the spectrophotometer a beam of light is transmitted through a bacterial suspension to a light sensitive detector. As bacterial number increases, less light will reach the detector. This change of light will register on the instrument’s scale as the percentage of transmission. Advantages: No incubation time required. Disadvantages: Cannot distinguish between live and dead bacteria. Requires a high concentration of bacteria (10 to 100 million cells/ml)

T URBIDITY

M ETABOLIC A CTIVITY As bacteria multiply in media, they produce certain products: Carbon dioxide Acids This method assumes that the amount of certain metabolic products is in direct proportion to the number of bacteria present.

D RY W EIGHT Bacteria or fungi in liquid media are centrifuged. Resulting cell pellet is weighed. Doesn’t distinguish live and dead cells.