Bacterial Cultivation

• Refers to the increase in the number of bacteria rather than in size

  bacterial growth
• This growth is affected by various factors such as optimum growth requirements, dynamics of growth, including the use of a medium that can be artificially prepared in the laboratory
• Bacterial growth requirement

  Nutritional and Environmental
• In bacteria, growth refers to the number, not in size. Basically, bacteria grow in number and do not grow in size
• three major nutritional needs for bacterial growth

  carbon, nitrogen, energy
• • Needed for the synthesis of cellular components
  • Represents almost 50% of the dry weight of the bacterium
  • could be from the carbon dioxide
  • Most of the normal flora in our body are heterotrophs

  Carbon
• Carbon Dioxide from the air

  autotrophs
• Organic compounds in the culture medium (glucose)

  heterotrophs
• • In the form of ATP: to perform metabolic and cellular functions
  • Light - Phototrophs
  • Chemotrophs - some are getting it from organic compounds and capable of converting them into energy


  Energy Source
• • Litotrophs - organic molecules (Fe2+)
  • Organotrophs - organic molecule
  • NADH reduced from NAD
  • FADH2 reduced from FAD


  Electron Source
• • Needed for the synthesis of proteins and nucleic acids
  • Makes up 14% of the dry weight of the bacteria
  • Free nitrogen from the air/atmosphere

  Example: those who are anaerobic, we need to add nitrogenous compounds

  • Nitrogenous compounds in the culture media (e.g., peptone, yeast, beef extract)

  Nitrogen
• • Bacterial cell is 70% water
  • Meaning, they are a sealed plate of moisture, so humidity level should be 70%.
  • In the laboratory, put a container of water inside the incubator that should be monitored regularly –check daily for molds, fungi that could grow that could be contaminants of our culture.

  Water/Moisture/Humidity
• Needed as co-factors in various metabolic process of the bacteria (e.g. Ca2+, Mg2+, Fe2+, Sulfates, Phosphates)

  Some bacteria requires smaller amounts of

  molecules such as:

  o phosphate - for nucleic acid

  o phospholipids - for cell membrane

  o sulfur - for protein synthesis

  
  

  Mineral Elements
• Mineral Element

  • Phosphate is for

  nucleic acids
• Mineral Element

  Phospholipid is for

  cell membrane
• Mineral Element

  • Sulfur is for

  protein synthesis
• Bacteria can tolerate salt concentration below 6%, however there are certain bacteria that can survive high salt concentration environment hence they’re called as HALOPHILIC or HALOPHILES (salt loving)


  Salt
• usually grow in MSA [7% sodium chloride]

  Staphylococcus spp.
• There bacteria that are very difficult to grow which we refer to as Fastidious (very difficult to grow) which requires special or additional requirements to grow in culture media
• Hematin is a degradation product of hemoglobin (RBC)

  X FACTOR (HEMIN/HEMATIN)
• • V is for Vitamin
  • Required by the Haemophilus spp. (adding blood to culture medium)

  V FACTOR (NICOTINAMINE ADENINE DINUCLEOTIDE OR NAD)
• There are 2 common types of culture media that has an X and V Factor (merong blood;Both contains blood, but CAP has lysed RBC; BAP

  has intact RBC

  Chocolate Agar Plate and Blood Agar Plate
• CAP has both X and V Factor

  ▪ Apply physical method such as heat that will activate the NADase; therefore NAD will be present = V factor is present
• BAP has only X Factor; there is no V factor in BAP

  ▪ since the RBC is intact, we have NADase is hydrolyzing the NAD (V factor); therefore, nawawalan ng V factor because it is degraded by the NADase
• Sources of Blood:

  • 5% defribinated sheep’s blood
  • Horse’s blood
  • Rabbit’s blood
  • Human blood – type O – non-specific inhibitor
• Bacteria that grow, live, and survive in the presence of oxygen

  Aerobe
• Absolutely requires oxygen to grown, live, and survive

  Micrococcus spp.,

  Mycobacterium spp.,

  Pseudomonas spp.,

  Neisseria spp., Brucella

  spp., Francisella spp.,

  Bordetella spp., Leptospira

  spp

  strict/obligate aerobe
• Bacteria that have the ability to grow, live and survive in small

  concentration of oxygen environment; hindi maarte

  Staphylococcus spp.,

  Streptococcus spp., Family

  Enterobacteriaceae

  facultative anaerobe
• Bacteria that prefers small concentration of oxygen environment approximately 2%- 10%

  • Usually, we have 18% of oxygen available in the environment
  • Campylobacter spp., Helicobacter spp., Arcobacter spp., and some Streptococcus spp.


  microaerophilic
• Bacteria that grow, live, and survive in the absence of oxygen

  anaerobe
• Absolutely do not require oxygen to grow, live, and survive

  Many Clostridium spp., such

  as Clostridium noyvi, most

  Bacteroides spp.,

  Fusobacterium spp.,

  Peptostreptococcus spp., and

  Porphyromonas spp.

  strict/obligate anaerobe
• Bacteria that do not require oxygen but may tolerate or withstand limited exposure to oxygen

  Some Clostridium spp., such

  as Clostridium perfringens,

  Bacteroides fragilis, most strains of Proprionibacterium

  and Lactobacillus


  aerotolerant anaerobe
• Bacteria that requires 5%-10% CO2 to grow

  • Usually, CO2 has 1-3% in the environment
  • NHACEK GROUP

  o Neisseria spp.

  o Haemophilus spp.

  o Aggregatibacter spp.

  o Cardiobacterium spp.

  o Eikenella spp.

  o Kingella spp.

  • Streptococcus pneumoniae

  capnophilic/capnophiles bacteria
• Most pathogenic bacteria would grow at temperature between 35-37°C, hence incubator in the laboratory is usually set and maintained within this temperature range for routine isolation of

  pathogens
• Optimally growing between 20-40 degrees Celsius


  Mesophilic
• Growing optimally between 0-20 degrees Celsius


  Psychrophilic/Cryophilic
• 50-60 degrees Celsius


  Thermophilic
• ▪ 80-110 degrees Celsius

  ▪ Spore - forming bacteria


  Hyperthermophilic/Extremely Thermophilic
• Wide range of temperature


  Eurithermophilic
• Narrow range of temperature


  Sternothermophilic