LO4 Bacterial Cell Cultures PDF

Learning Objective: 04 BACTERIAL CELL CULTURES  Cell Culture Technique Isolation and maintenance of cells in-vitro From tissues or organs derived from animals, microbes or plants success depends on number of factors; ✓ proper equipment ✓ sterile technique ✓ culture media a...

Learning Objective: 04 BACTERIAL CELL CULTURES  Cell Culture Technique Isolation and maintenance of cells in-vitro From tissues or organs derived from animals, microbes or plants success depends on number of factors; ✓ proper equipment ✓ sterile technique ✓ culture media and conditions ✓ appropriate protocols for handling  BACTERIAL CELL CULTURE Bacterial cell culture is the process by which bacterial cells are grown under controlled conditions, generally outside of their natural environment Why Culturing Bacteria? Isolation, detection, and differentiation of bacteria and other microbes that cause infectious disease To produce novel drugs (antibiotics) To produce biofertilizers  Basic Requirements to Maintain a Microbial Culture Sterile pure culture of cells-pure culture may originate from a single cell or single organism Appropriate aseptic techniques -to avoid contamination caused by harmful bacteria, viruses, or other pathogenic microorganisms Use of suitable conditions for optimal viable growth  Nutritional Requirements for Bacterial Growth Carbon and nitrogen are essential for bacterial growth Nitrogen sources – i.e. yeast extract, peptone (They not only contain nitrogen but also certain growth factors) Two categories according to Carbon source 1. Autotrophs - Requires water, inorganic salts and carbon dioxide (CO 2) for growth - Can synthesize essential organic metabolites from CO 2 2. Heterotrophs - Requires an organic source of carbon - Derive chemical energy by breaking down consumed organic molecules (sugars, proteins)  Autotrophs Synthesize food in the form of sugars –Photo-autotrophs Use sunlight as the energy source and manufacture food from inorganic raw materials – C Source – CO2 – Energy source - Sunlight –Chemo-autotrophs Use inorganic/organic substances as energy source and manufacture food from inorganic raw materials – C Source – CO2 – Energy source - Inorganic/ organic substance Heterotrophs Cannot produce its own food, instead take nutrition from other sources of organic carbon Derive chemical energy by breaking down consumed organic molecules (C Source) –Photo-heterotrophs Use sunlight as energy source and depend on organic matter already produced by other organisms for its nourishment C Source – Organic molecules – Energy source - Sunlight –Chemo-heterotrophs Use organic/inorganic energy source and absorb organic molecules as direct food/C source – C Source – Organic molecules – Energy source - Organic/inorganic substance  Oxygen Requirement Aerobes- Able to grow in the presence of atmospheric O2 Anaerobes- Able to grow in the absence of atmospheric O2 Obligate aerobes- Completely depend on atmospheric O2 for growth Obligate anaerobes- do not tolerate O2 and die in its presence Facultative anaerobes- Do not require O2 but do grow better in its presence Microaerophiles- Damaged by the normal atmospheric levels of O2 (21%) and require O2 levels between the range of 2% to 16% for growth  Culturing of Microorganisms Grow and harvest microorganisms Multiplying of micro-organisms under laboratory conditions-bioreactors Growth media, which can be either solid or liquid, provides the surface where micro-organisms grow Three main types 1. Batch Culture 2. Fed-batch Culture 3. Continuous Culture What is a bioreactor? An apparatus for growing organisms (yeast, bacteria, or animal cells) under controlled conditions. Used in industrial processes to produce pharmaceuticals, vaccines, or antibodies Also used to convert raw materials into useful byproducts such as in the bioconversion of corn into ethanol 1. Batch Culture / Closed System – A closed system where all nutrients are present at the start of the fermentation within a fixed volume – Nutrient supply is limited, most commonly used – Growth will continue until the medium is depleted of nutrients – Excessive build-up of toxic waste products generated by the microbes – Harvest is collected right before nutrients get depleted Disadvantages Requires very high mass of nutrients at the beginning Those nutrients can produce inhibitory substances Eg: Methanol, ethanol, acetic acid Growth rate is slower because the nutrient level declines with time 2. Fed-batch Culture/ Semi-open system A modification to batch fermentation Fresh medium or medium components are fed intermittently and the volume of the batch increases with time Controlled sequential additions of nutrients Advantages of fed-batch over batch culture are: Long-term synthesis of products Increasing the number of cells and thereby increasing the amount of product Greater efficiencies 3. Continuous Culture / Open System Open systems where fresh medium is continuously fed into the fermentation vessel, but the volume remains constant as spent medium and cells are removed at the same rate – Medium is refreshed regularly to replace the spent nutrients – Products and cells are continuously withdrawn – Maintain the cells in the exponential growth phase – Nutrient addition and waste removal can be controlled Disadvantages – More complex to set up – High risk of contamination Differences between batch, fed-batch & continuous culture  Culture Media Culture medium or growth medium is a liquid or solid gel designed to support the growth of microorganisms There are different types of media suitable for growing different types of cells Types of culture media 1. Based on nature/ consistency Liquid medium- To culture large quantity of microbes – Flasks – Large culture vessels-Fermenters Solid medium- To separate bacteria (isolate colonies), maintain stock cultures – Petri dishes Semi-Solid-To check motility of bacteria Agar- Complex polysaccharide/ gelatinous substance that is obtained from marine algae (red algae) (solidifying agent of culture media) 2. Based on components – Chemically defined / Synthetic media Exact concentration of every chemical is known - Minimal media – Non-defined media/ Chemically complex Exact concentration of every chemical is not known Contain natural ingredients (plant/ animal – Beef Extract agar, Yeast Extract Agar – Tryptic soy broth  3. Based on the Function A) General-purpose media-support the growth of most non-fastidious bacteria, primary isolation of microorganisms (Nutrient Agar) B) Selective media-suppress the growth of some microorganisms while allowing the growth of others (MacConkey Agar, Eosin Methylene Blue (EMB), Mannitol Salt Agar (MSA) C) Enrichment media-increase the relative concentration of certain microorganisms in the culture (broth) D) Differential/ indicator media-different bacteria can be recognized on the basis of their colony color (Mannitol salts agar, blood agar)  Microbial Control/ Sterilization The process that eliminates, removes, kills, or deactivates all living microorganisms present in a specified region, such as a surface or a volume of fluid Effectiveness depends on, – Type of micro-organism – Number of microorganisms present – Type and number of cracks and crevices in the instrument Two types – Physical Control – Chemical Control  Physical Control Radiation – UV Surface sterilization Cannot penetrate mucus, water, dust – X-ray, Gamma rays Filtration – Doesn’t destroy microorganisms completely – reduce microbial load – Pore size 10µm – 0.3 µm – For heat sensitive liquids Heat – Dry Heat – Moist Heat  Heat – Dry Heat Flaming – Inoculation loops, Inoculation needles Incineration – >500 0C – Biohazard waste discarding Oven Sterilization – 170 0C for 1 hr – 160 0C for 2 hrs – Glassware – Moist Heat Boiling – Max 100 0C – Surgical instruments Autoclaving – 121 0C , for 20 mins, 15 psi – Culture media  Chemical Control Require special handling Commonly used, – Glutaraldehyde – 2 to 4% for 10hrs – Formaldyhyde – 5% for 24hrs (Carcinogenic) – Gases – Ethylene oxide – Oxidizing agents – H2O2, O3 For medical devices  Aseptic Transfer Techniques Sub-culturing – the removal of the medium and transfer of cells/ colonies from the previous culture medium into fresh growth medium – Proper techniques should be used  Sterilization of Inoculation Needle and Loop Hold it to the hottest portion of the Bunsen burner flame working towards handle to loop – inner blue cone Keep until it becomes red hot Pass upper portion rapidly through the flame Cool for 10-20 secs  Alcohol Flaming of Forceps Hold the forceps horizontally Apply small amount of alcohol using a dropper Pass the tip through the flame Slowly open the forceps tips Allow alcohol to burn off Repeat the procedure for 3 times  Sterile Transfer to a Medium Obtain a bacterial culture Flame the inoculation loop and allow to cool Open the petri plate halfway and obtain the culture Mouth flame the test tube containing media Inoculate Again, mouth flame the test tube and close it Sterilize the loop Thank You!