nut3 bacterial cultivation

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

Microbiology Practical (Course I)

Mrs. Hibah Abdul-Wahab I. Abu-Sulaiman

Who will be her groom??!

Royal Dining Royal Atmosphere

Bacterial nutritional requirements. Composition of culture media. Categories of culture media. Solidifying agents. Types of media. Media Quality control. Culturing techniques. Condition of incubation.

1. Carbon Source:◦ Heterotrphs: from organic compounds such as:

a.a., polysaccharides, CHO, proteins and peptides.

◦ Autotrophs: from fixing inorganic carbon dioxide.

2. Energy Source:◦ Phototrophs: from sunlight.◦ Chemotrophs: from oxidation of chemicals.

3. Nitrogen: essential for a.a., nucleotide and vitamins synthesis.

4. Minerals: as cofactors in enzymatic reactions.◦ E.g., sodium, potassium, calcium, magnesium, iron…

etc.5. Growth factors:

◦ Amino acids, a.a.: bacteria possess proteinases.◦ Purines and pyrimidines: to be converted to

nucleotides.◦ Bacterial vitamins: for co-enzyme production.

6. Water: ◦ All metabolic functions of a bacterial cell are

performed in aqueous environment.◦ Distilled water

Tap water minerals and their reaction with peptones and meat extracts.

1. Water.2. Peptone: product of protein hydrolysis.

◦ From animal proteins: source of nitrogen.◦ From soya bean (plant) protein: source of

carbohydrates, CHO.

3. Meat extracts: as source of a.a. and other essential vitamins and elements.

4. Yeast extract: as a stimulant for bacterial growth in the media.

5. Mineral salts: for bacterial enzymes activity.6. Carbohydrates: as source of carbon and

energy.

Agar:◦ Is a complex polysaccharide (polymer of

galactose) that is extracted from the cell wall of seaweed (red algae).

◦ It has no nutritional value in agar cultures. Gelatin:

◦ A protein which is derived from collagen of skin and bone.

◦ It has no nutritional value in agar cultures.

Without agar

With agar

Consist of

Agar Slant Agar Deep Agar Plates

In broths, bacterial growth is indicated by a change in the appearance from clear to turbid (cloudy).

The more turbid, the….

Basal Media:◦ Simple media that will support the growth of most

bacteria that do not require special nutrient. E.g., Nutrient broth.

◦ Nutrient broth+ agar= Nutrient agar Enriched media:

◦ Culture media that are enriched with whole or lysed blood, serum, special extracts or nutrient to support the growth of those bacteria that cannot grow on the basal media.

◦ Nutrient agar+ blood= Blood agar◦ Blood agar+ heat= chocolate agar

Selective media:◦ Solid media which contain substances that inhibit the

growth of some bacteria and letting the others grow.

Enrichment media:◦ These are liquid media that are similar in function

to ...◦ The only difference is that…

Differential media:◦ Contains indicators that will differentiate one

organism from another Transport media:

◦ Contain substances that can prevent overgrowth of commensals and prevent bacteria from dying as a result of change in pH or enzyme action.

Media should be tested for:1. Sterility.2. Each medium must be tested with organisms

expected to grow or give a positive reaction as well as with organisms expected not to grow.

The media looks like this…

But when we’ll go over it, it’ll look like this ;)

Liquid Sample (or Broth) Swab

An agar

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

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.

Acidophiles: bacteria that grow in acidic pH. Neutrophiles: those that grow in between 4-

9. Alkalophiles: ones that grow in alkaline pH.

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%).