nobody answering my questions lol ,_,
okay I'm getting somewhere
so how to basic biotech is just read your notes and remember how to calculate everything
and cry.
You see many well separated colonies on your 16-streak plates. Describe a method you could use to confirm the purity of these separated colonies. [2m]
I won't even try writing notes because
actually I did write notes for LT1 (
Cells
Bacteria:
Flagella (surface structure) - Helps bacteria to swim towards nutrient-rich regions, light, oxygen-rich areas.
Pili (surface structure) - Exchange of genetic material between cells.
Fimbriae (surface structure) - Helps bacteria to adhere to host tissue/environment.
Glycocalyx - Protection from engulfment by white blood cells and harsh environmental conditions.
Plasmids - Carry genes that help the host cells be antibiotic resistant/be able to digest certain nutrients.
Nucleoid - Codes for genetic information of the bacteria.
Endospore - Resistant to numerous environmental conditions.
Animal/Plant:
Cell vacuoles (plant) - Maintain high internal water pressure, absorb water, aids in the support of the plant, store vital chemicals or waste products.
Nucleus - Stores the genetic material of the cell in the form of multiple linear chromosomes.
Smooth endoplasmic reticulum - Synthesis of lipids.
Rough endoplasmic reticulum - Synthesis of secreted proteins.
Mitochondria - Matrix contains enzymes for generating ATPs and mitochondrial DNA.
Chloroplasts - Thylakoids contain enzymes and chlorophyll for photosynthesis for glucose production.
Golgi body - Involved in modification, packaging and secretion of materials.
Bacteria/Animal/Plant:
Cell wall - Maintains characteristic shape of cell, prevents cell from bursting.
Cell membrane - Separates cell from environment, allows selected compounds to enter cells and waste to exit, allows proteins to attach to the surface of cells.
Ribosomes - Sites of protein synthesis.
Cytoplasm - Substance in the cell.
Culture of Bacteria
Aseptic Techniques:
Pure culture - Composed of only one strain of microorganism, easy to study the characteristics of the microorganism in detail, contaminants affect the growth of the microorganism of interest/alter their characteristics, any study (research/clinical) requires pure (homogeneous) strains as a start.
Container - Must be covered to prevent entrance of microorganisms on dust particles and on aerosols, cover should not be removed any longer than necessary, use the cover to shelter the container opening during the aseptic transfer, never set the cover down on a contaminated surface.
Growth medium/Container - Must be sterilised as soon as it is prepared.
Instruments - Must not touch contaminated surface, will transfer contaminants to pure culture.
Instruments/Fluids - Must first be sterilised if touching the inside of the container, the sterile medium or the culture, must be sterilised before returning to work area (inoculating loop), must be placed in disinfectant solution immediately after transferring cultures (pipettes), avoid creating aerosols.
16 Streak:
Inoculating loop
Bacteria:
Flagella (surface structure) - Helps bacteria to swim towards nutrient-rich regions, light, oxygen-rich areas.
Pili (surface structure) - Exchange of genetic material between cells.
Fimbriae (surface structure) - Helps bacteria to adhere to host tissue/environment.
Glycocalyx - Protection from engulfment by white blood cells and harsh environmental conditions.
Plasmids - Carry genes that help the host cells be antibiotic resistant/be able to digest certain nutrients.
Nucleoid - Codes for genetic information of the bacteria.
Endospore - Resistant to numerous environmental conditions.
Animal/Plant:
Cell vacuoles (plant) - Maintain high internal water pressure, absorb water, aids in the support of the plant, store vital chemicals or waste products.
Nucleus - Stores the genetic material of the cell in the form of multiple linear chromosomes.
Smooth endoplasmic reticulum - Synthesis of lipids.
Rough endoplasmic reticulum - Synthesis of secreted proteins.
Mitochondria - Matrix contains enzymes for generating ATPs and mitochondrial DNA.
Chloroplasts - Thylakoids contain enzymes and chlorophyll for photosynthesis for glucose production.
Golgi body - Involved in modification, packaging and secretion of materials.
Bacteria/Animal/Plant:
Cell wall - Maintains characteristic shape of cell, prevents cell from bursting.
Cell membrane - Separates cell from environment, allows selected compounds to enter cells and waste to exit, allows proteins to attach to the surface of cells.
Ribosomes - Sites of protein synthesis.
Cytoplasm - Substance in the cell.
Culture of Bacteria
Aseptic Techniques:
Pure culture - Composed of only one strain of microorganism, easy to study the characteristics of the microorganism in detail, contaminants affect the growth of the microorganism of interest/alter their characteristics, any study (research/clinical) requires pure (homogeneous) strains as a start.
Container - Must be covered to prevent entrance of microorganisms on dust particles and on aerosols, cover should not be removed any longer than necessary, use the cover to shelter the container opening during the aseptic transfer, never set the cover down on a contaminated surface.
Growth medium/Container - Must be sterilised as soon as it is prepared.
Instruments - Must not touch contaminated surface, will transfer contaminants to pure culture.
Instruments/Fluids - Must first be sterilised if touching the inside of the container, the sterile medium or the culture, must be sterilised before returning to work area (inoculating loop), must be placed in disinfectant solution immediately after transferring cultures (pipettes), avoid creating aerosols.
16 Streak:
Inoculating loop
Confirm culture purity - 1st round, 2nd round, etc.
Serial Dilution/Spread Plate/Colony Counting:
Calculation -
average colonies/0.1ml/10^-number of transfers
Unit - cfu/ml.
Standard form - _ x 10^_
Spread - 0.1ml.
Plate spreader - L-shaped glass rod.
30 to 300 colonies
1ml=1000µl
Growth Curve:
Lag Phase - Adapting to new environment, build up of cellular components for growth, cells increase in size but do not divide.
Log Phrase - Exponential cell growth, highest reproduction and metabolic rate.
Stationary Phase - Slow down in growth due to depletion of nutrients and increased waste products, death rate = growth rate.
Death Phase - Exponential cell death, death rate > growth rate.
Culture Media:
Defined medium - All components and their concentrations are known.
Complex medium - Contain some ingredients of unknown composition/concentration.
Characterisation of Bacteria
Cell Shapes:
Rod - Bacillus (coccobacilli (roundish), mycobacteria (long, thin), streptomycetes (moldlike, filamentous)).
Spherical - Coccus, (diplococci (in pairs), streptococci (in chains)).
Spiral - Spirillum.
Curved Rod - Vibrio.
Cell Wall:
Gram staining - Use a sterile loop to mix bacteria in water on a slide, air-dry, heat-fix, use crystal violet as primary stain (stains cells blue), iodine as mordant (increases affinity between primary stain and reactive substances in cell), alcohol as decolourising agent (good dehydrating agent) and safranin as counterstain (contrasts with primary stain), Gram +ve (cell wall is 90% peptidoglycan, high amounts of teichoic acids, dark blue), Gram -ve (cell wall is 10% peptidoglycan, outer membrane called lipo-polysaccharides, red)
KOH test - Rapid, suspend a loopful of culture into a drop of 3% potassium hydroxide, Gram -ve (cell will lyse causing its DNA to exude from the cell, can be detected by moving the loop up and down and a thin strand extending between the loop and the KOH emulsion will appear), Gram +ve (no strand of DNA will appear).
Penicillin - Binds with and inhibit the transpeptidases, preventing the cross-linking of the peptidoglycan, bacteria cell walls weakened, burst under osmotic pressure.
Kirby-Bauer - Filter disks impregnated with various antibiotics are placed on agar plate, antibiotics diffuse from disk into agar, establishing concentration gradient, incubate overnight, clear zones around disks where antibiotic inhibits bacterial growth.
Environmental Factors:
Acidophiles - pH 0 to pH 5.5.
Neutrophiles - pH 5.5 to pH 8.0.
Alkaliphiles - pH 8.0 to pH 11.5.
Thermophiles - 60ºC optimum temperature.
Mesophiles - 39ºC optimum temperature.
Psychrophiles - 4ºC optimum temperature.
Obligate aerobe - Need oxygen.
Facultative anaerobe - Prefer oxygen.
Strict anaerobe - Oxygen is toxic.
Microaerophile - <2% to 10% oxygen.
Aerotolerant anaerobe - Ignore oxygen.
Serial Dilution/Spread Plate/Colony Counting:
Calculation -
average colonies/0.1ml/10^-number of transfers
Unit - cfu/ml.
Standard form - _ x 10^_
Spread - 0.1ml.
Plate spreader - L-shaped glass rod.
30 to 300 colonies
1ml=1000µl
Growth Curve:
Lag Phase - Adapting to new environment, build up of cellular components for growth, cells increase in size but do not divide.
Log Phrase - Exponential cell growth, highest reproduction and metabolic rate.
Stationary Phase - Slow down in growth due to depletion of nutrients and increased waste products, death rate = growth rate.
Death Phase - Exponential cell death, death rate > growth rate.
Culture Media:
Defined medium - All components and their concentrations are known.
Complex medium - Contain some ingredients of unknown composition/concentration.
Characterisation of Bacteria
Cell Shapes:
Rod - Bacillus (coccobacilli (roundish), mycobacteria (long, thin), streptomycetes (moldlike, filamentous)).
Spherical - Coccus, (diplococci (in pairs), streptococci (in chains)).
Spiral - Spirillum.
Curved Rod - Vibrio.
Cell Wall:
Gram staining - Use a sterile loop to mix bacteria in water on a slide, air-dry, heat-fix, use crystal violet as primary stain (stains cells blue), iodine as mordant (increases affinity between primary stain and reactive substances in cell), alcohol as decolourising agent (good dehydrating agent) and safranin as counterstain (contrasts with primary stain), Gram +ve (cell wall is 90% peptidoglycan, high amounts of teichoic acids, dark blue), Gram -ve (cell wall is 10% peptidoglycan, outer membrane called lipo-polysaccharides, red)
KOH test - Rapid, suspend a loopful of culture into a drop of 3% potassium hydroxide, Gram -ve (cell will lyse causing its DNA to exude from the cell, can be detected by moving the loop up and down and a thin strand extending between the loop and the KOH emulsion will appear), Gram +ve (no strand of DNA will appear).
Penicillin - Binds with and inhibit the transpeptidases, preventing the cross-linking of the peptidoglycan, bacteria cell walls weakened, burst under osmotic pressure.
Kirby-Bauer - Filter disks impregnated with various antibiotics are placed on agar plate, antibiotics diffuse from disk into agar, establishing concentration gradient, incubate overnight, clear zones around disks where antibiotic inhibits bacterial growth.
Environmental Factors:
Acidophiles - pH 0 to pH 5.5.
Neutrophiles - pH 5.5 to pH 8.0.
Alkaliphiles - pH 8.0 to pH 11.5.
Thermophiles - 60ºC optimum temperature.
Mesophiles - 39ºC optimum temperature.
Psychrophiles - 4ºC optimum temperature.
Obligate aerobe - Need oxygen.
Facultative anaerobe - Prefer oxygen.
Strict anaerobe - Oxygen is toxic.
Microaerophile - <2% to 10% oxygen.
Aerotolerant anaerobe - Ignore oxygen.
Carbon/Energy Requirement:
Photoautotroph - Energy from the sun to break up CO2 molecules and make glucose.
Photoheterotroph - Use light energy to generate ATP, take in organic compounds from the environment.
Chemoautotroph - Synthesise their own organic molecules from the fixation of CO2, energy comes from the oxidation of inorganic molecules.
Chemoheterotroph - Consume pre-formed organic compounds as carbon source, utilises inorganic energy (chemolithoheterotroph) or utilises organic energy (chemoorganoheterotroph).
Biochemical Tests:
Oxidase (Cytochrome C) - Wet filter paper with TetraMethyl-p-PhenyleneDiamine (TMPD), use a sterile toothpick to streak bacteria on filter paper, cytochrome c +ve (strict aerobe, possess cytochrome oxidase, dark purple), cytochrome c -ve (does not possess cytochrome oxidase, colourless/light purple).
Catalase - Young cultures only, use disposable loop to apply bacteria on slide, add a drop of 3% hydrogen peroxide, +ve reaction (possess catalase, can respire with oxygen, strong: easily see bubbles, weak: use 10x objective microscope), -ve reaction (does not possess catalase, cannot respire with oxygen, no bubbles).
Starch hydrolysis - Inoculate starch agar plate (NA + 0.2% starch) with single streak, incubate at 37ºC for 2-3 days, flood plate with iodine solution, +ve reaction (possess amylase (exo-enzyme, degrades starch), clearing observed around growth area), -ve reaction (does not possess amylase, purple).
Industrial Microbiology
Enzymes:
Laundry detergents - Proteases, lipases, amylases (stain removal), cellulase (fabric softening, brightening), Bacillus licheniformis.
Syrup production, soft drinks, bakery, confectionary - amylase (starch to glucose), glucose isomerase (glucose to fructose).
Agriculture:
Nitrogen fixation - Conversion of atmospheric N2 to ammonia (NH3), usually via lightning, some bacteria have enzymes to convert N2 to ammonium salt (NH3+) and nitrate (NO3-), which can be absorbed by plants (most plants cannot use atmospheric N2).
Food:
Yogurt - Lactic acid bacteria (Lactococcus lactis), ferments lactose to produce lactic acids (turns milk into curd).
Cheese - Lactic acid bacteria (Lactococcus lactis), converts lactose into lactic acids (curding).
Wine/beer - Yeast, fermentation of fruit juice/malt grains.
Vinegar - Acetic acid bacteria (Gluconobacter, Acetobacter), partially oxidises ethanol to acetic acid.
Probiotics - The addition of microorganisms in the diet in order to provide health benefits beyond basic nutritive value, improve intestinal microbial balance and inhibit pathogens and toxin producing bacteria, prevent and treat pathogen-induced diarrhoea and urogenital infections, adjust/regulate immune response.
Photoautotroph - Energy from the sun to break up CO2 molecules and make glucose.
Photoheterotroph - Use light energy to generate ATP, take in organic compounds from the environment.
Chemoautotroph - Synthesise their own organic molecules from the fixation of CO2, energy comes from the oxidation of inorganic molecules.
Chemoheterotroph - Consume pre-formed organic compounds as carbon source, utilises inorganic energy (chemolithoheterotroph) or utilises organic energy (chemoorganoheterotroph).
Biochemical Tests:
Oxidase (Cytochrome C) - Wet filter paper with TetraMethyl-p-PhenyleneDiamine (TMPD), use a sterile toothpick to streak bacteria on filter paper, cytochrome c +ve (strict aerobe, possess cytochrome oxidase, dark purple), cytochrome c -ve (does not possess cytochrome oxidase, colourless/light purple).
Catalase - Young cultures only, use disposable loop to apply bacteria on slide, add a drop of 3% hydrogen peroxide, +ve reaction (possess catalase, can respire with oxygen, strong: easily see bubbles, weak: use 10x objective microscope), -ve reaction (does not possess catalase, cannot respire with oxygen, no bubbles).
Starch hydrolysis - Inoculate starch agar plate (NA + 0.2% starch) with single streak, incubate at 37ºC for 2-3 days, flood plate with iodine solution, +ve reaction (possess amylase (exo-enzyme, degrades starch), clearing observed around growth area), -ve reaction (does not possess amylase, purple).
Industrial Microbiology
Enzymes:
Laundry detergents - Proteases, lipases, amylases (stain removal), cellulase (fabric softening, brightening), Bacillus licheniformis.
Syrup production, soft drinks, bakery, confectionary - amylase (starch to glucose), glucose isomerase (glucose to fructose).
Agriculture:
Nitrogen fixation - Conversion of atmospheric N2 to ammonia (NH3), usually via lightning, some bacteria have enzymes to convert N2 to ammonium salt (NH3+) and nitrate (NO3-), which can be absorbed by plants (most plants cannot use atmospheric N2).
Food:
Yogurt - Lactic acid bacteria (Lactococcus lactis), ferments lactose to produce lactic acids (turns milk into curd).
Cheese - Lactic acid bacteria (Lactococcus lactis), converts lactose into lactic acids (curding).
Wine/beer - Yeast, fermentation of fruit juice/malt grains.
Vinegar - Acetic acid bacteria (Gluconobacter, Acetobacter), partially oxidises ethanol to acetic acid.
Probiotics - The addition of microorganisms in the diet in order to provide health benefits beyond basic nutritive value, improve intestinal microbial balance and inhibit pathogens and toxin producing bacteria, prevent and treat pathogen-induced diarrhoea and urogenital infections, adjust/regulate immune response.
I'll go for a break and come back soon.
gene tech and cell culture I will read accordingly to the study guides
YAY tomorrow got 4 hours to revise
I think microbial is the worst topic, so at least I got that outta the way
I go read bye.
answer for last year eoy
-lynn