Microbiology and Immunology

Dilution theory and techniques WORLD OF MICROBIOLOGY AND IMMUNOLOGY

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DIFFUSION•seeCELL MEMBRANE TRANSPORT

DIGEORGE SYNDROME•seeIMMUNODEFICIENCY

DISEASE SYNDROMES

DDilution theory and techniquesILUTION THEORY AND TECHNIQUES

Dilution allows the number of living bacteriato be determined

in suspensions that contain even very large numbers of bacteria.

The number of bacteria obtained by dilution of a culture

can involve growth of the living bacteria on a solid growth

source, the so-called dilution plating technique. The objective

of dilution plating is to have growth of the bacteria on the sur-

face of the medium in a form known as a colony. Theoretically

each colony arises from a single bacterium. So, a value called

the colony-forming unit can be obtained. The acceptable range

of colonies that needs to be present is between 30 and 300. If

there is less than 30 colonies, the sample has been diluted too

much and there is too a great variation in the number of

colonies in each milliliter (ml) of the dilution examined.

Confidence cannot be placed in the result. Conversely, if there

are more than 300 colonies, the over-crowded colonies cannot

be distinguished from one another.

To use an example, if a sample contained 100 living bac-

teria per ml, and if a single milliliter was added to the growth

medium, then upon incubation to allow the bacteria to grow

into colonies, there should be 100 colonies present. If, how-

ever, the sample contained 1,000 living bacteria per ml, then

plating a single ml onto the growth medium would produce far

too many colonies to count. What is needed in the second case

is an intervening step. Here, a volume is withdrawn from the

sample and added to a known volume of fluid. Typically either

one ml or 10 ml is withdrawn. These would then be added to

nine or 90 ml of fluid, respectively. The fluid used is usually

something known as a buffer, which is fluid that does not pro-

vide nutrients to the bacteria but does provide the ions needed

to maintain the bacteria in a healthy state. The original culture

would thus have been diluted by 10 times. Now, if a milliliter

of the diluted suspension was added to the growth medium, the

number of colonies should be one-tenth of 1,000 (= 100). The

number of colonies observed is then multiplied by the dilution

factor to yield the number of living bacteria in the original cul-

ture. In this example, 100 colonies multiplied by the dilution

factor of 10 yields 1,000 bacteria per ml of the original culture.

In practice, more than a single ten-fold dilution is

required to obtain a countable number of bacterial colonies.

Cultures routinely contain millions of living bacteria per mil-

liliter. So, a culture may need to be diluted millions of times.

This can be achieved in two ways. The first way is known as

serial dilution. An initial 10-times dilution would be prepared

as above. After making sure the bacteria are evenly dispersed

throughout, for example, 10 ml of buffer, one milliliter of the

dilution would be withdrawn and added to nine milliliters of

buffer. This would produce a 10-times dilution of the first dilu-

tion, or a 100-times dilution of the original culture. A milliliter

of the second dilution could be withdrawn and added to

another nine milliliters of buffer (1,000 dilution of the original

culture) and so on. Then, one milliliter of each dilution can be

added to separate plates of growth medium and the number of

colonies determined after incubation. Those plates that contain

between 30 and 300 colonies could be used to determine the

number of living bacteria in the original culture.

The other means of dilution involves diluting the sam-

ple by 100 times each time, instead of 10 times. Taking one

milliliter of culture or dilution and adding it to 99 ml of buffer

accomplish this. The advantage of this dilution scheme is that

dilution is obtained using fewer materials. However, the dilu-

tion steps can be so great that the countable range of 30-300 is

missed, necessitating a repeat of the entire procedure.

Another dilution method is termed the “most probable

number” method. Here, 10-fold dilutions of the sample are

made. Then, each of these dilutions is used to inoculate tubes

of growth medium. Each dilution is used to inoculate either a

set of three or five tubes. After incubation the number of tubes

that show growth are determined. Then, a chart is consulted

and the number of positive tubes in each set of each sample

dilution is used to determine the most probable number

(MPN) of bacteria per milliliter of the original culture.

See alsoAgar and agarose; Laboratory techniques in microbi-

ology; Qualitative and quantitative techniques in microbiology

DDinoflagellatesINOFLAGELLATES

Dinoflagellates are microorganismsthat are regarded as algae.

Their wide array of exotic shapes and, sometimes, armored

appearance is distinct from other algae. The closest microor-

ganism in appearance are the diatoms.

Dinoflagellates are single-celled organisms. There are

nearly 2000 known living species. Some are bacterial in size,

while the largest, Noctiluca, can be up to two millimeters in

size. This is large enough to be seen by the unaided eye.

Ninety per cent of all known dinoflagellates live in the

ocean, although freshwater species also exist. In fact, dinofla-

gellates have even been isolated from snow. In these environ-

ments, the organisms can exist as free-living and independent

forms, or can take up residence in another organism. A num-

ber of photosynthetic dinoflagellates inhabit sponges, corals,

jellyfish, and flatworms. The association is symbiotic. The

host provides a protective environment and the growth of the

dinoflagellates impart nutritive carbohydrates to the host.

As their name implies, flagella are present. Indeed, the

term dinoflagellate means whirling flagella. Typically, there

are two flagella. One of these circles around the body of the

cell, often lying in a groove called the cingulum. The other fla-

gellum sticks outward from the surface of the cell. Both fla-

gella are inserted into the dinoflagellate at the same point. The

arrangement of the flagella can cause the organism to move in

a spiral trajectory.

The complex appearance, relative to other algae and

bacteria, is carried onward to other aspects of dinoflagellate

behavior and growth. Some dinoflagellates feed on other

microorganisms, while others produce energy using photosyn-

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