Microbiology Flashcards
What do microorganisms include?
bacteria, fungi, protoctists and viruses
What do some bacteria and fungi do?
decompose dead organisms, releasing and recycling nutrients
What are some bacteria?
pathogens that cause disease in humans, crops and domestic animals, while others are harmless or beneficial.
How do bacteria reproduce?
asexually by binary fission and can do so very rapidly.
How many bacteria cells does the human body consist of?
one trillion cells
How many bacteria does the human gut contain?
approximately one hundred trillion bacteria from 500 to 1,000 different species.
What kingdom are bacteria in?
Prokaryote
How can bacteria be distinguished from eachother?
by their:
•Size
•Shape
•Staining characteristics
•Metabolic features
•Antigenic features
•Genetic features
What are the three bacteria shapes?
-Coccus (spherical) e.g. Staphylococcus, Streptococcus
–Bacillus (rod-shaped) e.g. Escherichia coli
–Spirillum (spiral/comma/corkscrew) e.g. Spirillum, Vibrio cholerae
What are the two metabolic features bacteria can be distinguished form eachother by?
-Autotrophic
-Photoautotrophic
What is photoautotrophic?
Either photosynthesis with chlorophyll as an e- donor, or alternatives using sulphur or hydrogen gas as an e- donor
What is autotrophic?
synthesise cell constituents using carbon dioxide as the carbon source
How can bacteria be distinguished from each other by their agentic features?
-an antigen is a molecule that causes the immune system to produce antibodies against it.
-These may be individual molecules or those on the surface of the bacterial cells.
What does the gram stain technique do?
classify bacteria (gram negative or gram positive)
What is the procedure for the gram stain technique?
-Fixation
-Application of crystal violet (purple dye)
-Application of Grams Iodine solution
-Alcohol wash (decolourisation) - differential stage
-Application of Safranin (a counter- stain)
What is the gram stain used by microbiologists to do?
distinguish between two types of bacteria; Gram-positive and Gram-negative
What are the different staining properties due to?
differences in the chemical composition of the cell walls.
What are the features of gram positive bacterial cell walls?
-Gram-positive bacteria have a thick peptidoglycan cell wall, but no outer lipopolysaccharide membrane.
-They therefore retain the initial crystal violet stain when washed with alcohol and appear purple under a microscope. Positive = Purple
What are the features of gram negative bacterial cell walls?
-Gram-negative bacteria have a thin peptidoglycan cell wall and an outer lipopolysaccharide membrane.
-When washed with alcohol, they lose this outer layer with the crystal violet stain.
-They are then able to take up the counter stain safranin and appear red under a microscope.
Why are some gram negative bacteria not susceptible to some antibiotics such as penicillin or lysosome (in tears)?
Due to the more complex cell wall
What are examples of gram positive bacteria?
Staphylococcus, Streptococcus
What is an example of gram negative bacteria?
Salmonella
How can bacteria reproduce?
Bacteria can reproduce rapidly through binary fission (division of bacteria) in a suitable environment, with division occurring every twenty minutes under optimal conditions.
What temperature are human pathogenic bacteria grown at?
37c
What temperature is bacteria grown in if it’s not pathogenic?
25c
What temperature do micro-organisms require for growth?
-Bacterial metabolism is enzyme-regulated, with most bacteria thriving between 25oC and 45oC.
-The optimum temperature for mammalian pathogens is around 37oC, the temperature of the human body
What nutrients do micro-organisms require for growth?
-In the laboratory, nutrients are supplied in nutrient media, such as nutrient agar or liquid broth.
-The carbon source is usually glucose, while nitrogen for amino acid and nucleic acid synthesis is provided as nitrate ions
What pH do micro-organisms require for growth?
Bacteria tend to favour slightly alkaline conditions, while most fungi thrive in neutral to slightly acidic environments.
What are obligate aerobes?
/can only survive and metabolise in the presence of oxygen.
-They cannot survive without it.
What are obligate anaerobes?
can only survive and metabolise in the absence of oxygen.
What are facultative anaerobes?
metabolise better in the presence of oxygen but can also survive and metabolise without it
Clostridium perfringens are obligate anaerobes that grow in wounds, producing toxins that cause gas gangrene. Symptoms include blisters under the skin, foul-smelling fluid and jaundice. Treatment can involve the use of a hyperbaric oxygen chamber with air pressure 2.5 x higher than atmospheric pressure.
How would this treatment work to improve the patient’s health?
Oxygen will inhibit metabolism & growth of C.perfringens
Patient’s immune system can kill any current bacteria
Fewer toxins produced – patient begins to improve
What is an example of an obligate anaerobe?
Clostridium tetani
What is an example of a facultative anaerobe?
Escherichia coli
What is an example of an obligate aerobe?
nitrobacter and nitrosomonas
What are at least two substrates that can be used to release energy in respiration?
-Glucose (enters glycolysis);
-fatty acids (to acetyl-CoÄ that enters the Krebs cycle);
-Glycerol (enters glycolysis as triose phosphate);
-amino acids (enter Krebs cycle)
Most of the energy released during respiration is used to synthesise ATP. How is the rest of the energy released?
Heat energy
Give the similarities and differences between aerobic and anaerobic respiration.
Similarities: both involve glycolysis (with a net of 2x ATP and 2x NADH/H+). Alcoholic fermentation produces CO2 and so does aerobic respiration.
Differences: Aerobic respiration uses 02 (final electron acceptor) but anaerobic does not.
Aerobic respiration has the link reaction. Krebs cycle and ETC (oxidative phosphorylation) inside mitochondria but anaerobic respiration does not.
What happens in the lag phase?
-No/little cell division.
-Intense metabolic activity such as enzyme/protein synthesis.
-Increased ATP production at mesosome (bacteria)
-Yeast Cells also rehydrating
What happens during the Log/Exponential Phase?
-Rapid increase in numbers, no limiting factors to growth.
-Cell division > death rate.
-Plenty of available glucose.
What happens during the stationary phase?
-Limiting factors prevent further growth of the population.
-E.g. competition for glucose.
-Carrying capacity has been reached.
What happens during the death (decline phase)?
-Limiting factors cause the population size to decrease.
-Death rate > cell division.
-This could be due to the build up of toxic waste, or no glucose left in the medium.
How would the description of a growth curve differ for a population of rabbits in a field?
Birth rate not cell division.
Lag could involve waiting for sexual maturity.
Limiting factors such as competition for mates.
What is a pathogen?
Microorganisms that act as parasites
What do aseptic techniques do?
keeps apparatus and equipment free of micro-organisms
What are the two main purpose of aseptic techniques?
-Preventing contamination of pure cultures by environmental microbes
-Preventing contamination of the environment by cultures being grown
How is contamination of pure cultures by environmental microbes prevented?
-Sterilise all media and equipment (e.g. inoculating loops) before use.
-Handle cultures carefully, flaming the neck of culture bottles before opening and closing.
-Use a lit Bunsen burner to create a convection current.
-Disinfect workbenches beforehand, such as with 3% Lysol.
How is contamination of the environment by cultures being grown prevented?
•Sterilise work surfaces before and after experiments using a disinfectant.
•Lift agar dish lid to no more than 45 degrees.
•Seal agar dishes with adhesive tape, but not all the way around.
•Flame the neck of the culture bottle without placing the cap on the work surface.
What should you do when inoculating an agar dish?
-Pass the metal inoculating loop through a flame until red hot, then allow it to cool.
-Hold the bacterial culture bottle in one hand; remove the cap with the little finger of the other hand without placing it down.
-Flame the neck of the culture bottle for 2-3 seconds and dip the inoculating loop into the bacterial culture.
-Lift the petri dish lid to 45 degrees, allowing entry of the inoculating loop, and streak the agar with the bacterial culture.
-Secure the petri dish with adhesive tape, but do not seal it completely to avoid anaerobic conditions that could promote pathogenic growth.
-Incubate at a suitable temperature (25 or 37oC) for 24-48 hours.
-Sterilise all equipment after use.
What are the two ways equipment should be sterilised before and after use?
-Sterilisation using an Autoclave (Glassware and Metal Equipment)
-Sterilisation using Gamma Irradiation (Plastic Equipment)
How is equipment sterilised using an autoclave?
-In a laboratory the preferred method of sterilisation is an autoclave. This sealed vessel sterilises equipment by:
•Sealing it in an autoclave bag
•Heating it to 121°C in steam (above boiling point)
•Applying high pressure for 15 minutes
How is equipment sterilised using Gamma Irradiation?
-Plastic equipment is often sterilised with gamma irradiation before use.
-After use, plastic equipment can be disposed of in a biohazard waste bin or sterilised in an autoclave
How can microorganism populations in liquid culture be measured?
by counting cells directly or indirectly
What is a total cell count?
Living and dead cells in a bacterial sample
What is a way of directly counting cells? (total cell count)
-For example, a haemocytometer, a microscope slide with a grid-engraved rectangular chamber, can be used to calculate the number of microbes in a sample.
-The chamber has a known depth, allowing the number of cells in a specific volume to be counted.
-A coverslip is placed over the chamber and the slide is observed under a microscope to count the cells.
What is Serial Dilution?
total viable count technique
Why is serial dilution used?
It is not possible to count an entire population of microorganisms directly. Instead, cells in a small culture sample are counted.
What does serial dilution do?
-assumes a single bacterial cell will reproduce asexually to form a visible colony on an agar plate after incubation. -The number of colonies therefore represents the number of bacteria in the original sample
What is the procedure of serial dilution?
-Total viable cell count is the number of living bacteria in a given volume (e.g. 1 cm3)
-1cm3 of original culture is added to 9cm3 of sterile water (or sterile culture medium) and gently mixed, to make a 1:10 dilution;
-1cm3 of this 1:10 dilution is then added to 9cm3 of sterile water (or sterile culture medium) and gently mixed, to make a 1:100 dilution;
-Repeat procedure to make 1:1000, 1;10,000, 1;100,000 dilutions (to a minimum of 10-5 dilutions);
-Transfer 1cm3 of each dilution onto agar plates (spread out with sterile spreader) and incubate plates for 24-48 hours at 25-300C
each bacterium forms a visible colony;
-Count number of distinct colonies on agar plates where no merging and then multiply number of colonies by dilution factor = number of bacteria in 1 cm3
What inaccuracies may occur when carrying out a serial dilution if the original bacterial culture is under diluted?
If dilution is insufficient then colonies might merge, referred to as “clumping” and counting may be inaccurate resulting in an under-estimate of cell numbers
What inaccuracies may occur when carrying out a serial dilution if the original bacteria culture is over diluted?
If dilution is too great then there will be too few colonies on each plate to count to be statistically sound (leading to inaccuracies)
When performing serial dilution calculations what is it crucial to not?
-the volume of the original starting culture (usually 1 cm3) and the volume of each dilution spread onto the agar plates.
-This ensures accurate calculation of the number of bacteria in the original culture
How are cells indirectly counted?
by measuring the turbidity (cloudiness) of the culture gives an indirect measure of growth
What is the the procedure of indirectly counting cells?
-Turbidimetry involves using a colorimeter to measure the turbidity (cloudiness) of a culture as cell numbers increase.
-Bacterial population measurements are obtained by finding the suspension’s absorbance value and referencing a standard graph of light absorbance plotted against the number of bacterial cells.
-This results in a total cell count, as the colorimeter cannot differentiate between living and dead cells.
What is a prokaryote?
The kingdom that bacteria are found in
What is gram-positive?
A bacterium which stains purple (due to crystal violet)
What is gram-negative?
A bacterium which stains red (due to safranin)
What is binary fission?
Process by which bacteria divide
What is an aseptic technique?
Process by which apparatus and equipment are kept free of microorganisms
What is an autoclave?
A sealed vessel in which glass and metal equipment are heated to 121oC at high pressure to sterilise them
What is a Haemocytometer?
Microscope slide used to calculate the number of microbes in a sample
What is serial dilution?
A method of calculating viable count using repeated dilution of bacterial culture, assuming one colony will arise form one bacterial cell
What is under-dilution?
This causes colonies to merge (referred to as clumping), making counting inaccurate
What is over-dilution?
This leads to there being too few colonies on each plate, making the results not statistically sound
What is Turbidimetry?
A method of indirectly counting cells using a colorimeter (giving a total cell count)
