Basic microbiological techniques

Types of growth patterns in broth

Clear

Turbid (cloudy)

Flocculent

Pellicle

Sediment

Liquid media is?

Water-based solutions that do not solidify at room temperatures above freezing and tend to flow freely when the container is tilted

• LIKE NUTRIENT BROTH

• CONTAINS PEPTONE AND BEEF EXTRACT DISSOLVED IN WATER ****** (essential nutrients for the growth of bacteria)

semisolid media is?

it exhibits a clot-like consistency at room temperature. They contain a certain amount of solidifying agent (agar or gelatin) that thickens them but does not produce a firm substrate.

• Such media are used to determine the motility(movement) of bacteria

how is bacteria innoculated in the semisolid media?

The microorganism is stabbed below the

surface of the agar and the pattern of growth

is examined after incubation ⇒ The growth of

non-motile organisms will be restricted to the

original stab line whereas motile organisms

will spread through the agar and produce a

diffuse cloudiness that is referred to as the

"Christmas Tree" patterns of growth.•

what is solid media, how is it used, what is the solidifying agent typically used

Solid media provide a firm surface on which cells can

form discrete colonies. They are advantageous for

isolating and subculturing bacteria and fungi. They

contain a solidifying agent, agar which is the most

effective and widely used agent in solid media.

Agar is? What are its solidifying characteristics?

is a complex sugar isolated from the red algae Gelidium. It is solid at room temperature and liquefies (melts) at the boiling temperature of water. Once liquefied, agar does not resolidify until it cools to 42 degrees celcius.

Agar can be? (innoculated) Why, and what does the agar do for microorganisms

Agar can be inoculated and poured in liquid form at

45 degrees celcius to 50 degrees celcius C so that it will not harm the microbes or

the handler.

Agar is flexible and moldable, and it

provides a basic framework to hold moisture and

nutrients, though it is not itself a digestible nutrient

for most microorganisms.

Features of a good media culture (solid medium)

Completely sterile

Smooth, even for the inoculation of bacteria

solid at room temperature

does not re-solidify quickly

does not harm the microbes or the handler

does not prevent the growth of microorganisms

not a digestible nutrient for the microorganism

How can we use streak plate method?

A small droplet of the sample is spread over the surface of the medium according to a pattern that gradually thins out the sample and spatially separates the cells over several sections of the surface of the agar.

Loop dilution or Pour plate method

The sample is inoculated serially into a series of cooled

But still agar tubes so as to dilute the number of cells in

each successive tube in the series. Inoculated tubes are

then poured into a petri dish dish and are

allowed to solidify

Spread plate method

A small volume (0.1 ml to 1.0 ml) of the diluted sample is

pipetted onto the surface of the medium and spread

around evenly by a sterile spreading tool (sometimes

called a ‘hockey stick’). The cells are pushed into separate

areas on the surface so that they can form individual

colonies

How do we achieve dilution?

To reduce amount of bacteria transported to the next section, allowing for the dilution of bacteria sample and the growth of well-separated colonies.

How do we ensure aseptic environment on a work surface?

Surface sterilisation:

Clean work areas by spraying 70% ethanol onto them and allow time for disinfection

• Ethanol disrupts the cell membranes of the microorganisms, leading to death

How do we ensure aseptic environment on the apparatus or materials used?

i.e.

Use autoclave

• For sterilising nutrient media and other apparatus

Or

Metal inoculating loop

• Sterilise over a flame before use

THESE MEASURES ENSURES,

• Material and apparatus used are free from contaminating organisms

How do we ensure aseptic environment in the surrounding air?

Work near a LUMINOUS flame (for safety, can be seen) of a bunsen burner

• Draws up air current upwards (forming a barrier)

• This prevents the entry of contaminating microorganisms onto the work surface

Can also tilt petri-dish at an angle of 45 degrees to ensure that contamination is minimised as you keep most of the lid as a barrier hence it limits the gap through which contaminating microorganisms can reach the agar.

Why should molten agar be maintained between 45 degrees celcius to 50 degrees celcius before pouring into a sterile petri dish?

• So it can be inoculated and poured in liquid form at that temperature

• will not hurt the microbes or the handler

examples of solidifying agents?

agar/gelatin

Why do we want to isolate bacteria cells?

If we separate an individual bacteria cell from other cells and is provided adequate space on a nutrient surface, it will grow into a discrete mound of cells called a COLONY.