Bacteria and other microbes have particular requirements for growth

When they reside in and on our bodies or in the environment, they harvest their food from us or from the environment

When we grow bacteria in lab, we are essentially creating a captive environment for bacteria – like a bacteria zoo. So we must provide the bacteria we grow in lab with all of the materials that they need to grow

In this lab we learn about different types of media that are used to grow bacteria. Some types of media will grow just about any type of bacteria and the others are more selective and only grow specific types of bacteria

There are generalized media, like (Nutrient agar)that will grow many different types of microbes.

This media is the type most often used to culture bacteria

culture medium that allows the growth types of organisms,

while inhibiting the growth of other organisms

EMB (Eosin Methylene Blue) dyes inhibit Gram (+) bacteria selects for Gram (-) bacteria

Example:

Differentiates between different organisms growing on the same plate Example: Blood agar

culture medium that includes ingredients, such as chemical indicators , that produce observable differences between species of bacteria

This media is differential because:

Certain bacteria produce enzymes (hemolysins…) that act on the red cells to produce either:

Beta hemolysis: Enzymes lyse the blood cells completely, producing a clear area around the colony.

Alpha hemolysis: Incomplete hemolysis produces a greenish discoloration around the colony

Gamma hemolysis: No effect on the red cells.

There are also (selective)

and

(differential)media

MacConkey’s is both a selective & differential media.

1. MacConkey’s is selective media because it inhibits the

growth of some organisms [Gram positive bacteria].

2. MacConkey’s is differential media - “lactose fermenters” bacteria will grow in red colonies while” non-lactose fermenters” will be colorless and clear.

So if there are colonies of bacteria growing on MacConkey’s, it’s understood that they are Gram- If those colonies are colorless, they are not lactose fermenters If the colonies have a pinkish appearance, they are lactose fermenters

left: no lactose fermentation

right: lactose fermentation

MacConkey Agar

FORMS OF CULTURE MEDIA

broth liquid medium

most common growth media for microorganisms are ( nutrient broths ( liquid nutrient medium

Solid media commonly contain 1.5% agar per weight to solidify the liquid media.

After sterilization, the media is poured into sterile Petrie plates.

Agar is liquefies at 100 C and solidifies at 40 C

General Media:

Nutrient Broth and Nutrient Agar

or After autoclaving the media(in tube) for 20 minutes, the tubes are placed in a slanted position to allow the agar to solidify. These tubes are called slants

Slant tubes:

are tubes containing a nutrient medium plus a solidifying agent, (agar-agar. The medium has been allowed to solidify at an angle in order to get a flat inoculating surface

Agar Plate:

are sterile petri plates that are filled with a melted sterile agar medium

Microorganisms grow on the surface of agar plates and slants

140 Micro

Lab 4: Isolation of microbes from

different environments

Isolation of micro organisms from various environments

1. Isolation –separating one species from another.

2. Inoculation – introduction of a sample into a container of media to produce a culture of observable growth.

3. Incubation – conditions that allow growth eg., temperature ,humidity etc..

4. Inspection.

5. Identification.

Environmental Sample Normal Flora Samples

Surface samples are normally taken using sterile swabs

Applying oral sample to surface of agar

Petri plates with media

Soil

Rotten fruit

Yoghurt

Mouth swab etc

Incubator……

Bacteria Fungi

Yoghurt

Mouth

Hand

Isolation of Bacteria

• Put a drop of diluted yoghurt on the solid media plate of bacteria . • Incubate at 37 °C for 1 day.

• Take some saliva with the help of a cotton swab. • Inoculate it on the media and incubate.

•Touch the surface of the solid media plate . • Incubate 37 °C for 1 day. .

Agar plates are stored upside down to prevent condensation.

These plates of bacteria will be incubated at 37° C for 24 hours and then stored at refrigerator until next week when you will observe for results.

Air

Soil

fruit

Isolation of Fungi

• Expose the prepared plate of solid media in air for 5 min • Close the lid and incubate at 28 °C. • After 2-5 days fungus observe for grow

• Sprinkle a pinch of soil on the solid media plate • Close the lid and incubate at 28 °C • After 2-3 days fungus observe for growth.

• Clean the rotten part of fruit with alcohol • Cut a piece and inoculate it on the media plate •Then incubate ,after 2-5 days fungus will grow

These plates of fungus will be incubated straight at 28° C for 24 hours and then stored at refrigerator until next week when you will observe for results.

Bacteria Fungi

الشهرانيشروق .أ