Bacterial cultivation, sterilization, and disinfection

• Bacterial growth refers to the increase in the number of bacteria rather than in size
• Bacterial growth


  • It 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 requirements

  • Nutritional
  • Environmental
• Nutritional requirements

  • Carbon
  • Nitrogen
  • Energy
• Carbon


  Needed for the synthesis of cellular components/constituents, represents almost 50% of the dry weight of the bacterium
• Carbon source


  • Carbon dioxide from the air (autotrophs)
  • Organic compounds in the culture media (glucose) (heterotrophs)
  • most of normal flora are heterotrophs
• Energy source


  • Light (phototrophs)
  • Chemical energy from organic compounds (chemotrophs)
• Electron source


  • Inorganic molecule (Fe2+) (lithotrophs)
  • Organic molecule (organotrophs)
• Nitrogen


  Needed for the synthesis of proteins and nucleic acids, makes up 14% of the dry weight of the bacteria
• Nitrogen source


  • Free nitrogen from the air/atmosphere
  • Nitrogenous compounds in the culture media (e.g., peptone, yeast, beef extract)
• Water/moisture/humidity


  Bacterial cell is 70% water, humidity level should be 70%
• Mineral elements

  • Ca2+
  • Mg2+
  • Fe2+
  • Sulfates
  • Phosphates
• Salt


  Bacteria can tolerate salt concentration below 6%, some bacteria are halophilic and can survive high salt concentration environment hence they're called halophilic
• Examples of halophilic bacteria

  • Staphylococcus spp. - usually grow in MSA [7% sodium chloride]
  • Enterococcus spp.
  • Vibrio spp. (except V. cholerae and V. mimicus) - up to 10% salt concentration can be tolerated
  • Bacillus spp.

  
• Fastidious bacteria


  Require special or additional requirements to grow in culture media
• X factor


  Hemin/Hematin, a degradation product of hemoglobin (RBC)
• V factor


  Nicotinamide Adenine Dinucleotide (NAD), required by Haemophilus spp.
• Culture media with X and V factors

  • Chocolate Agar Plate
  • Blood Agar Plate
• Chocolate Agar Plate


  Contains lysed RBC, has both X and V factors
• Blood Agar Plate


  Contains intact RBC, has only X factor (no V factor)
• Sources of blood


  • 5% defibrinated sheep's blood
  • Horse's blood
  • Rabbit's blood
  • Human blood - type O
• Gaseous requirements

  • Aerobe - Bacteria that grow, live, and survive in the presence of oxygen
  • Anaerobe - Bacteria that grow, live, and survive in the absence of oxygen
• Aerobic bacteria

  • Strict/obligate aerobe - Absolutely requires oxygen to grown, live, and survive
  • Facultative anaerobe - Bacteria that have the ability to grow, live and survive in small concentration of oxygen environment; hindi maarte
  • Microaerophilic - Bacteria that prefers small concentration of oxygen environment approximately 2%- 10%
• Anaerobic bacteria


  • Strict/obligate anaerobe - Absolutely do not require oxygen to grow, live, and survive
  • Aerotolerant anaerobe - Bacteria that do not require oxygen but may tolerate or withstand limited exposure to oxygen
  • capnophiles - Bacteria that requires 5%-10% CO2 to grow - Usually, CO2 has 1-3% in the environment
• Capnophilic/capnophiles/capnophilic bacteria

  • Neisseria spp.
  • Haemophilus spp.
  • Aggregatibacter spp.
  • Cardiobacterium spp.
  • Eikenella spp.
  • Kingella spp.
  • Streptococcus pneumoniae
• Temperature requirements- 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


  • Mesophilic - Optimally growing between 20-40 degrees Celsius
  • Psychrophilic/cryophilic - Growing optimally between 0-20 degrees Celsius
  • Thermophilic - 50-60 degrees Celsius
  • Hyperthermophilic/extremely thermophilic - 80-110 degrees Celsius - Spore-forming bacteria
  • Eurithermophilic - Wide range of temperature
  • Sternothermophilic - Narrow range of temperature
• pH requirements- most pathogenic bacteria can grow in a neutral or slightly alkaline environment (pH 7.0 – 7.5), hence most culture media used in routine isolation of pathogens is adjusted to this pH range


  • Acidophilic – Acid loving bacteria (e.g. e.g. Lactobacillus acidophilus)
  • Alkaliphilic – Alkali loving bacteria (e.g. Gardnerella vaginalis)
  • High osmotic pressure (osmophilic) – Osmophilic Bacteria (Archaebacteria spp.)
• Bacterial growth phase- refers to the stages of bacterial growth.


  • Lag phase - no cell division is happening; cellular activity: synthesis of proteins and DNA is already happening
  • Logarithmic growth phase - the number of generations per hour is called growth rate. ▪ The phase where bacteria is the most metabolically active; susceptible to anti-microbial agents ▪ May cell division na = increase in number
  • Stationary or plateau phase - rate of cell division = rate of cell deaths
  • Death (decline) phase - mas maraming namamatay kaysa sa viable
• Generation time/doubling time


  Time required for 1 cell to divide into 2 cells
• The generation time of bacteria can be from 20 minutes (E.coli) up to 24 hours (Mycobacterium tuberculosis)
• Lag phase


  Bacteria are adjusting to the environment, no cell division is happening, cellular activity like synthesis of proteins and DNA is already happening

  
• Logarithmic growth phase


  Rapid generation of bacteria, the number of generations per hour is called growth rate, bacteria are the most metabolically active and susceptible to antimicrobial agents
• Stationary or plateau phase


  Nutrients are depleted, toxins are produced that are harmful to microorganisms, rate of cell division = rate of cell deaths
• Death (decline) phase


  No more bacterial growth, more cells are dying than viable
• Growth rate

  • Lag phase: 0
  • Logarithmic: +
  • Stationary: 0
  • Decline: -
• Culture media


  An artificial preparation in the laboratory which contains basic foundation of nutrients and a solidifying agent (if needed) to support the growth of microorganisms
• Terminologies


  • Culture (noun: growth of microorganism, verb: to grow or to cultivate microorganism)
  • Inoculate/plant/cultivate (introducing the microorganism to the culture media)
  • Transplant/subculture (subsequent transfer of microorganisms from one culture media to another)
• Classification of culture media (according to composition)

  • Synthetic/chemically defined
  • Non-synthetic/non-chemically defined/complex
  • Tissue culture media
• Classification of culture media (according to physical state or consistency)

  • Liquid
  • Semi-solid
  • Solid
• Classification of culture media (according to manner of dispensing/formation)

  • Plated
  • Tubed
  • Bottled