3.2 Culture in Anaerobic (Anoxic) Chambers

Question 1

Nutritional Requirements

Answer 1

• > Macronutrients
• > Micronutrients
• > Growth factor

Question 2

Macronutrients

Answer 2

• larger amounts required
• water, carbohydrates, proteins / peptides, lipids (fats)

Carbon (Carbohydrates, sugars, cellular building blocks)

Nitrogen (proteins, nucleic acids)

Sulphur (amino acids (cysteine, methionine), vitamins)

Phosphorus (nucleic acids and phospholipids)

Metals:

• Potassium / Magnesium / Calcium
• required for enzyme activity, membrand and cell wall stability

Question 3

Micronutrients

Answer 3

• > required in small amounts, but critical
• > Excess can be toxic
• > Metals (trace elements) - e.g. manganese (component / cofactor in key enzymes) or iron

Iron:

• required in greater amounts than other trace elements
• major role in cellular respiration
• often in short supply, competition with other organisms
• Bacteria secrete siderophores
• important role particular in pathogenic bacteria

e.g. Myobacterim tuberculosis - secrets the siderophores myobactin

Question 4

Siderophores

Answer 4

iron-chelating compounds that bring ferric iron from the external environment into the cytoplasm

Question 5

growth factor

Answer 5

• organic compounds like vitamins, amino acids, purines and pyrimidines
• required in small amounts
• requirements do vary between microorganisms
• most commonly required:
  Vitamin B and Biotin

Question 6

Laboratory culture of microorganisms

Answer 6

• > culture (nutrient) medium - used to grow specific microbes in the lab
• > Buffers added to laboratory culture media to maintain pH - metabolic reactions consume and produce acidic and basic substances
• > Liquid (broth) or solid (broth containing agar) depending on purpose
• > often in shaking incubator to maintain oxygen levels

Sterile conditions:
requires sterilisation of culture medium

Question 7

Different Types of Culture media

Answer 7

1. Chemically defined medium
   * chemical composition known
2. Complex medium
   * nutrients at unknown concentration and in excess
   * contain quite often sigests derived from animals, plants and yeast

Question 8

Different types of culture media can be used as

Answer 8

1. Selective
   Contains compounds that selectively inhibits growth of some microbes to select specific group of microorganisms from a sample
2. Differential
   Chemical components differentiate between specific types of microbes typically used in clinical microbiology to differentiate bacteria.
   e.g. blood agar differentiateshemolytic from non-hemolytic organisms
3. Enrichment
   Supplemented with particular nutrients to increase numbers of specific kinds of bacteria

Question 9

Agar plates

Answer 9

-> Agar added to produce solid media:
unbranded polysaccharide obtained from the cell walls of some species of red algae
-> solid culture medium in petri dish
-> cells immobilised on solid media
-> allows streaking for isolation single cells
-> single cells grow and multiply into visible colonies

=> colony size, shape, texture and colour can be used in identification

Question 10

Unit of measure - colony forming units (CFU) per ml or mg

Answer 10

-

Question 11

many prokaryotes are still uncultured

Answer 11

retrieval analysis of ribosomal RNA genes from microorganisms in environmental samples have shown that many are phylogenetic distinct but as yet many uncultures prokaryotes exist in nature

Question 12

Viable but non-culturable state (VBNC)

Answer 12

• > microorganisms with a state of very low metabolic activity
• > cell do not divide, but are alive and have the ability to become culturable once resuscitated under certain conditions
• > often in response to stress (nutrient limitation, extreme temperatures, toxic, chemicals)
• > survival strategy
• > seen in variety of bacteria including pathogens