MICROBIAL CULTURING TECHNIQUES

AIM

To learn serial dilution method for enumeration of bacteria and various plating techniques to
obtain pure culture

INTRODUCTION

Various methods are used to obtain pure culture of bacteria or for enumeration of bacteria.
Procedures include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-
plating and (3) spread-plating to enumerate viable bacterial colonies.

The streak-plate procedure is designed to isolate pure cultures of bacteria, or colonies, from
mixed populations by simple mechanical separation. Single colonies are comprised of
millions of cells growing in a cluster on or within an agar plate. A colony, unlike a single cell,
is visible to the naked eye. In theory, all the cells in a colony are derived from a single
bacterium initially deposited on the plate and thus are referred to as a clone, or cluster of
genetically identical cells.

Bacteria exist in a variety of shapes and sizes. For example, individual Escherichia coli cells are
rod-shaped with an average length of 2 μm and width of 0.5 μm while Streptococcus cells are
spherical with an average diameter of 1 μm. Some bacteria (such as E. coli) exist as single cells
while others form distinct patterns of association. Streptococcus, for instance, grow in pairs or
form chains or clusters of cells. It is generally assumed that a single colony arises from a single
cell undergoing binary fission; however, this assumption is not true for those bacteria that
naturally exist as pairs, chains, or clusters or that divides by other mechanisms. Alternatively, if
too many bacteria are plated, then overlap of cells may occur and increase the probability of two
or more bacteria giving rise to what appears to be a single colony. To avoid these complications
when describing or enumerating bacterial cultures growing on a solid medium, colonies are
referred to as colony forming units (cfu).

With the streak-plate procedure, a mixture of cells is spread over the surface of a semi-solid,
agar-based nutrient medium in a Petri dish such that fewer and fewer bacterial cells are deposited
at widely separated points on the surface of the medium and, following incubation, develop into
colonies. The quadrant method for isolating single colonies from a mixture of cells will be
described here.

A serial dilution is the step-wise dilution of a substance in solution. Usually the dilution factor at
each step is constant, resulting in a geometric progression of the concentration in a logarithmic
fashion. A ten-fold serial dilution could be 1 M, 0.1 M, 0.01 M, 0.001 M... Serial dilutions are
used to accurately create highly-diluted solutions as well. A culture of microbes can be diluted in
the same fashion. For a ten-fold dilution on a 1 mL scale, vials are filled with 900 microliters of
water or media, and 100 microliters of the stock microbial solution are serially transferred, with
thorough mixing after every dilution step. The dilution of microbes is very important to get to
microbes diluted enough to count on a spread plate
Spread plates are simply microbes spread on a media plate. Microbes are in a solution, and can
be diluted. They are then transferred to a petri dish with media specific for the growth of the
microbe of interest. The solution is then spread uniformly through a number of possible means,
the most popular is the use of sterile glass beads that are shook on top of the media, spreading the
microbe-containing liquid evenly on the plate. Also common is the use of a bent-glass rod, often
referred to as a hockey stick, due to its similar shape. The glass rod is sterilized and used to
spread the microbe-containing liquid uniformly on the plate.

Pour Plate method often is used to count the number of microorganisms in a mixed sample,
which is added to a molten agar medium prior to its solidification. The process results in colonies
uniformly distributed throughout the solid medium when the appropriate sample dilution is
plated. This technique is used to perform viable plate counts, in which the total number of colony
forming units within the agar and on surface of the agar on a single plate is enumerated. Viable
plate counts provide scientists a standardized means to generate growth curves, to calculate the
concentration of cells in the tube from which the sample was plated, and to investigate the effect
of various environments or growth conditions on bacterial cell survival or growth rate.

PROCEDURE

Sterilize all instruments, solutions, and media prior to using them for plating procedures. Clean
work area with disinfectant to minimize possible contamination.

Streak-plating can be accomplished with a number of different instruments. A metal loop can be
re-used multiple times and is utilized for streak-plating routine laboratory strains. Label around
the edge of the bottom (not the lid) of an agar plate with at least your name, the date, the type of
growth medium, and the type of organism to be plated on the medium. The sample from which
the streak-plate will be inoculated could be either a suspension of cells in broth or an existing
colony from another agar plate. To begin, it is recommended that only one sample be used to
inoculate a single plate. To save time and materials, a single plate can be used for multiple
samples but only once you become proficient at streaking one sample on a plate.

For the first quadrant, the sample is picked up and spread over about one-quarter of the surface
of the medium using a rapid, smooth, back-and-forth motion. Lift the bottom half of an inverted
plate from the bench. Move the loop, stick, or toothpick back and forth many times across the
agar surface from the rim to the center of the plate. Turn the Petri dish 90 ° to streak the second
quadrant then third and fourth also.

Preparing serial dilutions is necessary if the number of cells in the sample exceeds the capacity
of the agar plate, in which the statistically significant range is 30 to 300 cfu. If there are more
than 300 cfu on a plate, then the colonies will be crowded and overlapping. Sample volume to be
plated should be between 0.1 and 1.0 ml. In pour plating measured sample in added in molten
agar, mixed thoroughly and poured in empty sterile petridish. Allowed to solidify. Incubated in
inverted position at respective time and temperature.

This technique typically is used to separate microorganisms contained within a small sample
volume, which is spread over the surface of an agar plate, resulting in the formation of discrete
colonies distributed evenly across the agar surface when the appropriate concentration of cells is
plated. With your right hand, hold the spreader gently on the surface of the agar and gradually
spread the sample evenly over the entire plate. Move the spreader back and forth across the plate
as the turntable is spinning. Allow the sample to absorb thoroughly (at least 5 minutes) before
inverting the plate for incubation.

RESULT

Learned serial dilution method for enumeration of bacteria and various plating techniques to
obtain pure culture.