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How to culture organisms
You have to give them what they need and what they like. It's that simple
Bacteria and food
They need a lot of food because they multiply quickly. No food = death, unless they are spores
Are formed during harsh environments. It's a signal to convert the organism into sport in order to survive.
Colony forming units. The cells are actually clones of the original cell
Microbial growth and results
- Growth - Increase in a population of microbes
- Results - An aggregation of cells arising from single parent cell
Nutrients: Chemical and Energy Reqs.
Major Nutrients - Carbon, energy, and electrons
Carbon nutrient groups
- Usually come from a source like beef extracts.
- Self feeding organisms. They make their own food like green plants and photosynthetic org. as for microbes algae and cyanobacteria.
- Carbon nutrient groups
- Depend on preformed organic material. Need other source of food, can't make their own.
- Carbon nutrient groups
They use chemical reactions to produce energy required for growth. Like we do with metabolism
Use the sun as their energy source
- Defined - Shows exactly the amounts for the exactly that is known.
- Undefined - Mcconkeys is undefined
Four basic groups of organisms
Carbon dioxide, Organic compounds, and chemical compounds
All they need is carbon dioxide mixed with light or chemical compounds
Preformed organic compounds. Mixed with light of pchemical compunds
T/F: Oxygen is a toxic biological molecule to all life forms.
True cuz oxygen utilization makes hyperactive free radicals that are extremely toxic. The free radicals break down cell walls, cell membranes and cause mutations in DNA. That is why you need the oxygen protective triad otherwise we would not be able to use oxygen and evolution would not have occured.
- Produced by metabolic action.
- Since oxygen makes hyperactive free radicals you have use hyperbolic oxygen to treat gangrene, wounds that do not heal etc.
Four Toxic Forms of Oxygen
- Singlet oxygen
- Superoxide radicals
- peroxide anion
- Hydroxyl radical
Bacteria that Oxygen affects
- Facultative anaerobes
- Aerotolerant anaerobes
- Have all 3 of the oxygen protective triad and can survive and grow in oxygen.
- Need to have 21% oxygen present
- Organism related: Pseudomonas
- Don't have any of the oxygen protective triad so they die in the presence of oxygen and cannot grow.
- Organism related: Clostridium
- Can grow with our without oxygen, they usually have at least 1 or 2 of the oxygen protective triad. Prefers to grow in oxygen cuz ATP production is better.
- Organism related: S. Aureus
- Typically have 1 of the oxygen protective triad. Not killed at the presence of oxygen. They can only tolerate/withstand oxygen not used for growth. Because they have inductive superoxide desmotase
- Oxygen is lethal @ high concentrations
- Organism Related: Bacteroides
- Can only grow under reduced oxygen 5-10% and increase CO2 8-10%.
- Organism Related: Campylobacter
Used for pp9 on 4-19 notes about aerobes to microaerophiles
- Gets recycled from amino acids and nucleotides
- Cyanobacteria can fix nitrogen and convert nitrogen from the atmosphere
- If too low = activity slows and membranes become rigid and fragile
- It too high = proteins are denatured and deactivated and membranes become too fluid
Four categories of microbes based on temperature ranges for growth
Psychrophiles and Temperature
- Love low temp
- Growth: -5 to 20°C
Mesophiles and Temperature
- Love medium temp
- Growth: 15 to 45°C
- These are most likely to infect humans because of our body temp is the same
Thermophiles and Temperature
- Love high temp
- Growth: 45 to 80°C
Hyperthermophiles and Temperature
- Love super hot temp like boiling water and stuff
- Growth: 65 to 105°C
- Is a Psychrotrophic bacteria. They are really mesophiles but can adapt to psychrophiles that is why they grow in cold environment.
- Clinical significance of Listeria: Septic abortion, neonatal sepsis, sepsis and septic shock, fetal death
- Organisms are sensitive to changes in acidity
- Neutrophiles grow best in a narrow range around neutral pH
- Acidophiles grow best in acidic habitats
- Alkalinophiles live in alkaline soils and water
Physical effects of water
- All organisms require water
- Used in many metabolic reactions
- Dehydration is the leading cause of death
- Endospores and cysts are the exception to dehydration
- Part of dehydration, difference between pickling and curing.
- In fact we use this when there is a high salt conent.
- Can also be used to make ham and jelly
- Pressure is exerted on a semipermeable membrane by a solution containing solutes that cannot freely cross membrane.
- Restricts organisms to certain environments
Have lower solute concentrations
Have greater solute concenrtations
S. Aureus and salt tolerance
It is salt tolerant and not a halophile
Halobacter salinarum and salt
- 12-25% salt tolerant = halophile.
- Can be found in salt marshes, ocean, and saltered sea/dead sea
- Vibrio species/ocean.
- Vibrio cholera
- Vibrio vulunificus
- Transmitted by fecal contamination
- Can affect human body because theya re facultative and not obligate.
- Which means they don't need salt to start growing
Contaminated shell fish
- High molecular weight polysaccharide
- This is the adaptive response found in facultative halophiles.
How to distinguish between obligate and facultative halophoes
- They are distinguished by the range of salt.
- The obligate need 15-25% salt to grow while the facultative need much smaller salt concentrations and can start at 0%
Look it up online
Use environment to grow in the same place and exploit it to their own advantage
Usually are between 2 or more organisms. Grow together in the same environment so they grow much faster and the effect is greater than what is expected
How to get an appropriate specimen. On written
- 1. Should contain viable organisms.
- - Protozoa are the exception cuz they do not have to be viable in order to be ID. They are the only preserved media.
- 2. Free from contamination
- 3. Specimen must be representative of te infectious process
- 4. Must be taken from the infection site and must also be free of org from neighboring sites
- 5. Processed ASAP or stored under conditions to preserve viability
- 6. Place in container to preserve growth or viability
- 7. Procedure for sampling must be appropriate
- 8.Taken in quantities adequate to permit work up
- 9. Label appropriately, including patients name, source, specimen etc.
- 10. Send to the lab in timely fashion
- Take bacteria and put it on something that will make it grow so proper nutrients are needed.
- Viruses, chlamydia need to be alive because the cannot be grown in artificial medium
When to culture for isolation?
For viruses and bacteria only, sometimes fungi
How are the five I's relavent?
Patient comes in to hospital and they are ill and they want it diagnosed and treated. You collect the specimen and process in lab which then becomes the basis of CNS. Have to isolate org, then Id by biochemistry, then do the susceptibilit of it. more on voice?
Standard Plate Count/ Serial Dilution
Was not designed for ID of org but to count how many bacteria is present
Describe streak for isolation as done clinically with adequate detail to document isolation.
Involves flaming and cooling of loop every time you streak. You drag 4 times and the 5th time 2 squiggles in the middle. The idea is to perform serial dilutions over the entire plate.
The most accepted and typical method to ??? (on voice 5-6-15)
Quadrant streak isolation is the answer.
Types of Culture Media
- Defined media
- Complex media
- Selective media
- Differential media
- Anaerobic media
- Transport media
- Every single known ingredient is established as well as the concentrations, volume, proportions are all established.
- Everything is established about the medium
Vast majority of these are used clinically
Mcconkeys, manitol salt agar (MSA), EMB are selective because the allow only certain bacteria to grow
Selective and differential for fecal coliforms in mcconkeys and EMB
Such as E.Coli
Why is MSA selective?
- Because it has 5% salt and it is inhibitoy for most bacteria other than S. Aureus.
- S. Aureus will grow while others wont
Why is Mcconkeys inhibitory?
- It has inhibitory crystal violet and will inhibit gram - bacteria as well as isolate them. Ex. E.Coli cuz it's a fecal coliform and is gram -
- Ferments lactose and turns it pink
Why is EMB selective and differential?
- Fecal coliforms have distinct representation on it like iridescent, blue black colonies w/ or w/o metallic sheen.
- Inhibits growth of gram +, favors growth of gram - and also gives ID because it has media that is utilized for these bacteria. This is why it is selective and differential.
- Vigorous fermentation of lactose and glucose will produce the above results with color etc.
The reactions are specific for the organism that is what makes it differential since it can tell which org is present based on its documented reaction
Specimen DNA, to transfer the bacteria in order to preserve it so it gets to the lab in a viable form for it to be examined.
Media used for fungal growth?
Sabourads dextrose agar because it's acidic and fungi prefer acidic conditions. Selective but not differential
- Generalize but are not specific
- Show hemolysis which is the destruction of red blood cells.
- Beta hemolysis
- Gamma hemolysis
- Alpha hemolysis
Partial grading of blood
ID group B streptococci based on their formation of a substance. Like a bow tie
A gram + coccus is shown to be beta hemolytic and gives a + CAMP reaction?
- Asnwer is: Streptococcus agalactiae because it's a group B.
- or Streptococcus pyogenes
Group B which causes strept throat
A gram + spore forming rod is shown in reverse CAMP
Clostridium perfringenese the enzymes produced by this literally digest skin cells that allow bacteria to persist and infect
Clinical Relevance of CAMP
- Related clinically because group b strept. should be screened for anyone contemplating pregnancy, both male and female.
- Male especially if they are donating sperm because it will transmit.
Pregnant Females Screening and CAMP relavence
- All pregnant females need to be screened for group B strept. Generally treated in the last trimester. Her partner should also be treated and tested because you can reinfect.
- This can cause sepsis since 40% of neonatal sepsis and meningitis is cause by ecoli and streptococcus galactia?
Special Culture Techniques
- Animal and cell culture
- Low-oxygen culture
- Enrichment culture
Animal and Cell Culture
Used for chlamydia and viruses
- Grow low oxygen microaerophiles.
- Bacteria that need very tiny oxygen amounts to grow.
A patient has diarrhea for 2 or 3 days, very watery stools and reports drinking raw milk. Campylobacter suspected. What environment would you provide to allow isolation of campylobacter?
- A. Aerobic
- B. Anearobic
- C. Microaerophilic
- D.None of the above
- Answer is C. Microaerophilic
- Refrigeration - Stores for short periods of time
- Deep-Freezing - Stores for years
- Lyophilization - Stores for decades
- Time required for bacterial cell to grow and divide.
- For humans it's 9 months
Microbial Grow and Clinical Importance
- They rapidly multiply, since they divide so rapidly even small numbers can lead to full virus infections.
- # of bacteria determine the virulence.
- High virulent org. need fewer # of bacteria to cause disease.
Virulence and Ex.
- Ability to cause disease measured by # required.
- Ex1. E. Coli OK157 causes hemolytic uremia which produces ~10-100 CFU so they are highly virulent
- Ex2. Salmonela has a infectious dose of 10^6 through 10^9 CFU to cause infection is it is less virulent than E. Coli
- During this phase they are preparing to divide and is a period of intense metabolic activity, not to be interpreted as being inactive or dormant.
- Time spent in lag phase depends on what environment it came from. Rich = less time in lag phase.
Log (exponential) Phase
Phase of most active growth and they are dividing in their maximum rate, it's linear and it's the only part of the curve that is associated with linear increasing.
Stationary Phase (Batch culture only)
- Continuous culture doesn't have this stage. It's synchronized.
- The bacteria are dieing as fast as they are produced so they = a steady state.
- Should not be confused with absence of growth or them stopping dividing.
Death (decline) Phase
- Continuous culture doesn't have a death phase.
- Linear decrease in #'s (exponential), and is happening because of exhaustion of nutrients and accumulation of waste products which are inhibitory.
- All bacteria are in the same stage of growth. Simultaneous division.
- They are multiplying as rapidly as possible because they have abundant nutrients and the inflow matches the outflow.
- Same rate of inflow and outflow which maintains the same growth.
Wash out of bacteria in culture
The bacteria numbers decrease significantly and there is no more culture left because of the inflow and the outflow
- There is no accumulation of cell death or waste products because they all go into the outflow.
- The material from the inflow and the outflow is the same and when that happens all the growth is synchronized and because of that they are in the exponential rate of growth.
Continuous Culture Advantages and Disadvantages
- It allows you to study physiological and adaptive responses in an organism
- The body is not a continuous culture so you can't predict how it will react unlike the controlled culture.
What is similar to a continuous culture?
Biofilms because as along as you have a solid substrate and nutrients you ahvre accumulation of bacteria
- Form of communication that bacteria use to talk to each other.
- If you stop this they wouldn't be able to talk to each other and then you can break up the biofilm essentially. Now you can control the biolfilm growth and formation
Serial Dilutions and Viable Plate Counts
- Part of the direct method.
- The dilution series is relevant because you can't count them all and that is why you do a series since if you do not know how many # of bacteria are present. That is why you cannot do 1 dilution you need a series.
Why do we use a 1 micro-meter loop and not an ordinary loop that is metal?
- The loop is calibrated because we have a known value which was 1 micro-meter.
- Relevant in UTI definition because we are looking for bacteria isolation that is = or greater than 10^5 CFU/ml and that is why you need a clean catch or a mid stream.
- Part of Direct Methods
- on voice
- Part of direct method
- You look for micro-colonies that means the bacteria is viable and if they are single cell that means they are dead.
There is a plate with 200 bacteria on it and it's a 10^-6 dilution. What is the original concentration since the dilution was made?
What are the acceptable #'s when doing a standard plate count to warrant estimation of growth?
- 30-300 are the acceptable numbers.
- If below 30 or above 300 they are no acceptable
- Part of Direct Method
- You can label cells coming through and then easily count them individually
Direct Methods of Measuring Microbial Reproduction
- Serial dilution and viable plate counts
- Membrane filtration
- Microscopic counts
- Electronic counters
Indirect Methods of Measuring Microbial Reproduction
- Metabolic Activity
- Dry Weight
- Part of the Indirect Method
- Shows if they are viable but doesn't say (rest on voice)
- Part of the Indirect Method
- You have to dry the material and then you have to dry it to the point where the is no change in dry weight at all (no water)
- Can't tell if dead or alive
Turbidity (Optical Density)
- Part of the Indirect Method
- Principle is if you have a lot of bacteria the bacteria will block the light and you wont have the light going through.
- How many bacteria are present in the mixture. If high OD then you have more bacteria and if you have low OD then you have less bacteria.
- Doesn't say if they are dead or alive because they still impede the passage of light that is why it is indirect.
- OD reading are linear up to 1 OD. Reading that are greater than 1 are not reliable.
% Transmittance can be converted to OD
- This is the way to get around OD, you can also dilute the sample till it falls into the curve of the 1 OD
The vast majority of bacteria have not been cultured. It is estimated that only 3-5% have actually been cultured.
T/F: Gene Replacement Therapy is No Longer a Myth?
Procedure of Human Gene Therapy
- Take gene and insert it into a specific virus that doesn't infect humans and use it as a shuttle.
- Cells from the target tissue are removed from the patient.
- The cells are grown in large numbers in tissue culture plates
- Then transfered back to patient
- Use virus as a shuttle to transfer the new gene into the cell to infect the cells and then you put the cells back into the patient
- 5 carbon sugars
- A & G = purine
- C, T, & U = pyramidine
How are DNA strands positioned?
They are anti parallel cuz the leading and lagging strands are in different positions.
Leading DNA Strands
- This strand is right side up
- Information is being received by this strand and goes from 5' --> 3'
- It is also a continuous strand
Lagging DNA Strand
- This strand is upside down
- Discontinuous cuz the orientation is not correct and goes from 3' --> 5'
# of hydrogen bonds between purines and pyramadines
- 2 hydrogen bonds between A and T
- 3 hydrogen bonds between G and C
Relevance of hydrogen bonds between purines and pyramidines
- Cuz the thermal stability of the DNA molecules is determined by it's G (guanine) and C (cytozine) ratio/content
- The higher the content the more heat stable the DNA is.
- Thermophiles and Hyperthermophiles will have the higest G and C content/ratio cuz they need it in order to survive in those conditions.
Flow of Information in Genetic Coding
DNA to RNA to Proteins.
- DNA to RNA
- Copying it word for word otherwise shits gets fucked up
RNA to Protein
Exceptions to the Flow of Information in Genetic Coding
- HIV, retroviruses
- Retroviruses use RNA to make DNA by using the enzyme called reverse transcriptase.This can be used to control replication of virus like HIV by inhibiting the reverse transcriptase
- Highly Aggressive Retrovital Therapy
- Used to treat HIV/AIDS
- DNA copying itself.
- When copying it makes 4 strands, 2 new and 2 old.
- Goes through cycles
How DNA Replication Cycles Work
- With each cycle 50% of DNA represents new synthesized DNA and the other half represents old DNA.
- Cycle 1: 50%
- Cycle 2: 25%
- Total: 50 + 25 = 75%
T/F: Definitive ID of Microbes in a mixed sample requires isolation in pure culture to ID by DNA probes
False, cuz once DNA probes bind, it only binds to the specific sequence which was obtained or present in the organism from which the probe was made.
Clinical Applications of DNA
- DNA Probes:
- Probe binding is based on complementary base pairing (A & T and G & C) and recognizes organism with that same sequence.
- Greatest % of binding is associated with the DNA obtained from the same organism
Features of DNA Probe
- The sequence is known. Must be shown to be present in all strains of the same species.
- Must not be subject to mutation or variation
- Must be stable
- Must be unique to the organism and not present in related strains or species
Why would a DNA probe be clinically useful?
- Highly specific and reproducible.
- Cost Effective
- Time saving
- Provides information that facilitates effective patient monitoring.