Second year Lab Medicine (Females)1433- 1434 A.H. / 2012- 2013

Microbiology Practical (Course I)

Mrs. Hibah Abdul-Wahab I. Abu-Sulaiman

Last week the bacteria was watching us as if we are…

Bacteria:

The media looks like this…But when we’ll go over it, it’ll look like this ;)

So let us…

After eating…

To know what they’re thinking about, you need to…?!

Learn how to read their personalities

Objective

• Culturing techniques.• Condition of incubation.• Reading culture plates.• Preparing bacterial smears.• Staining using Gram Stain.

Culturing Techniques

Liquid Sample (or Broth) Swab

An agar

Aseptic Techniques

Work area disinfection

Flaming loops and needles

Petri- plate inoculation

Condition of Incubation

• Temperature.• Hydrogen ion concentration (pH).• Gaseous requirements.

1. Temperature

• Too high temperature will denature bacterial ezymes. While too low temperature….

• Types of bacteria according to their growth temperature:1. Psychrophiles: bacteria that grow between 0-20oC.2. Mesophiles: those that grow between 21oC and

50oC.3. Thermophiles: the ones that grow between 50oC

and 100oC.

2. Hydrogen ion concentration (pH)

• Acidophiles: bacteria that grow in acidic pH.• Neutrophiles: those that grow in between 4-9.• Alkalophiles: ones that grow in alkaline pH.

3. Gaseous requirements

Bacteria differ in their need to molecular oxygen for growth:

• Obligatory (strict)aerobes: require free oxygen for growth.

• Obligatory (strict) anaerobes: grow only in the absence of free oxygen.

• Facultative anaerobes: multiply either in the presence or absence of free oxygen.

• Carboxyphilic (capnophilic): these bacteria require raised level of carbon dioxide (5-10%).

Gas Generating Jar

Reading the plates

1. Hemolysis2. Size.3. Form or margin.4. Elevation.5. Color.6. Consistency.7. Pigment.8. Odor.

Reading the plates

1. Hemolysis: – is a reaction caused by enzymatic or toxin activity of bacteria,

observed in the media immediately surrounding or underneath the colony.

– Proper technique requires passing of bright light through the bottom of the plate (transillumination) to determine wether the organism is hemolytic.

1. α-hemolysis: is partial lysing of erythrocytes in a BAP around and under the colony that results in a green discoloration of the medium.

2. β-hemolysis: is compelete clearing of erythrocytes in a BAP around or under the colonies because of the complete lysis of RBCs.

– Organisms that are α- hemolytic or β-hemolytic on BAP usually show a green coloration around the colony on CAP.

Hemolysis

α- Hemolysis β- Hemolysis

Reading the plates (Cont. …)

2. Size: colonies are described as large, medium, small or pinpoint.

– Gram positive bacteria, in general, produce smaller colonies than gram negative bacteria.

3. Form or margin: described as smooth, filamentous, rough or irregular.

3. Elevation: it should be determined by tilting the culture plate

and looking at the side of the colony. – Elevation may be raised, convex or dome, flat,

umbulicate(depressed center) or umbonate (raised center).

Reading the plates (Cont. …)

4. Color: colonies may be white, gray or yellow.

5. Pigment: – pigment production is an inherent characteristic

of a specific organism confined generally to the colony.

6. odor.

Pigmentation

Preparing Bacterial Smear

Staining Using Gram’s Stain (GS)

Thanks a lot