Topic 10 Flashcards
What are the physical requirements for microbial growth?
- Temperature
- pH
- Osmotic pressure
What are the chemical requirements for microbial growth?
- C source
- N source
- S source
- P source
- Organic growth factors
- Trace elements
- O2
What is the maximum growth temperature?
50 degrees Celcius
What is the optimum growth temperature?
approx 37 degrees Celcius
What is the minimum growth temperature?
approx 4 degrees Celcius
What temperature does Listeria monocytogenes grow at?
Low temperatures but growth is slowed
What plays a role in gastric ulcers?
Helicobacterpylori
What phile is Helicobacterpylori
Acidophile
What phile is Bacteria?
Neutrophile
What phile is Fungi?
Outside and inside Neutrophile
What pH can Helicobacter pylori withstand?
Low pH conditions
What happens in isotonic solution?
No water movement
What happens in hypertonic solution?
- Water exits cell
ex: salt curing process
Carbon (Chemical Requirements)
- For structural organic molecules
- Energy source
Nitrogen (Chemical Requirements)
In A.As, proteins
Sulfur (Chemical Requirements)
- In A.As, thiamine, biotin
- 4% dry weight of cell
Phosphorus (Chemical Requirements)
- In DNA, RNA, ATP, and phospholipids (membranes)
- 4% dry weight of cell
Organic Growth Factors (Chemical Requirements)
- Organic compounds the organism cannot synthesize on its own
- Usually not manufactured by the cell, must be obtained from environment
- Ex: vitamins, A.A, nucleotides
Trace Elements (Chemical Requirements)
- Inorganic elements required in small amounts, mostly for specific enzymes
- Iron, Copper, Zinc, Magnesium, Calcium, etc
What are the oxygen requirements?
It may vary with the organism
What does oxygen do to energy generation?
It makes it more efficient however O2 can be lethal to some microbes
What are obligate aerobes?
They require oxygen for survival and growth
What are obligate anaerobes?
They cannot tolerate oxygen and cease to grow (and/or die) in the presence of oxygen
What are facultative anaerobes?
They can survive in aerobic and anaerobic conditions but prefer to grow in the presence of oxygen
What type of medium do you observe aerobic and anaerobic growth in?
Thioglycolate broth medium
What oxygen can cause a highly destructive tissue infection?
Clostridium perfringens, an obligate anaerobe
What are the two anaerobic culture methods?
- “GasPak” anaerobic system (contains reducing media)
- Anaerobic glove box
How do microbes grow in nature?
Often grow as biofilms
How are microbes grown in the lab?
May be grown as mixed cultures or pure cultures
What does liquid media (broth) contain?
A.A, salts, and nutrients that microbes need to grow
What is liquid media (broth) used for?
To culture bacteria and yeast (and protists)
What can agar be added to?
Nearly added liquid media - used to culture bacteria, yeast, mould
How are microbes solidified?
Using agar
What is agar?
A carbohydrate extracted from seaweed
What is a colony?
When one cell multiplies
What can colony morphology tell us?
The # of different species based on colour (same colour might be a different species though)
Why is streaking done?
You dilute a sample so that a single bacterium will grow as a colony
What are the bacteria within a colony like?
They are clones (genetically identical)
Explains what happens by the 3rd and 4th streaks?
As you go back and forth you are spreading less and less bacteria (you are diluting the amount of bacteria)
Hopefully by the 3rd and 4th streaks you will have individual cells
How can you grow a colony?
Isolate a pure genetic sample on solid media if it is diluted so that a single bacterium will grow a colony
Why do you culture viruses?
- To study virus particles
- Vaccine production
How do we culture viruses?
- Viruses are obligate intracellular parasites (they can only grow in cells)
Therefore we must culture them in cells and not on dish plates
Where are bacteriophages grown?
On bacterial lawns
What is a plaque?
A hole that a virus has destroyed all that bacteria (happens as the virus replicates)
Do bacteriophages cause problems for humans?
No
What can we grow in egg cultures?
Animal viruses
How can you grow animal viruses?
May be grown in cell culture
What are the easiest cells to grow?
Cancerous cells (because they are non-differentiated (ex: not nerve or muscle cells) and they continuously divide rapidly
How can you use cells to make a virus grow?
Inject the cells with virus and have the virus grow
Are cell lines maintained?
Continuous cell lines may be maintained indefinitely (these cells lines are ‘immortal’ i.e tumor cells)
How are cancer cells different from other cells?
They can divide even if they are touching each other
Cancer cells and immortality
Some cancer cells are immortal (turn on genes) they do not die, they keep dividing
What is chemically define media?
The chemical composition is precisely known
What is an example of chemically defined media?
Minimal media
- contains only the bare essentials
- only certain species can grow
- often used to study genetic mutants
What is a type of non-selective media?
General or complex media
What does general or complex media encourage?
The growth of a range of microbes
What is complex media?
You are taking things and mixing things together but you don’t know the exact portion of things
Describe enriched media (2)
- Non-selective
- Contains additional factors needed to grow microbes with particular needs
Describe selective media
Only supports the growth of specific microbes
What type of cells does selective media allow to grow?
Gram (+) but not gram (-) cells
How many species grow with selective media?
One species
What grows on nonselective media
Everything grows
What is an example of selective media?
Pseudomonas Isolation Agar
- E.coli cannot grow
- Pseudomonas aeruginosa can grow
What conditions does fungi prefer?
Acidic
What temperature does fungi and bacteria like?
Fungi grow at lower temperatures (25) whereas bacteria like higher temperatures (37), bacteria and fungi compete with each other
Example of selective media (terms of bacteria or fungi)
pH of media may allow bacteria or fungi to grow
What pH does bacteria like?
pH 7
what pH does fungi like?
pH 6
Why do different species have a different appearance?
It is due to the characteristics of the medium (differential media)
How are X-gal and lactose related?
Anything that is capable of breaking down lactose can break down X-gal, it will break down and change colour
What is a type of differential media?
Media containing X-gal
How do blue colonies form?
When species/strains that can metabolize lactose (and X-gal) grow
What is the difference between non differential and differential media
Non-differential media: everything looks the same
Differential media: Species show differing reactions
What does EMB stand for?
Eosin methylene blue
What type of media is Eosin ethylene blue (EMB)?
Differential media
What does EMB media do?
Provides a colour indicator distinguishing between organisms that ferment lactose (eg. E. Coli) and those that do not (eg. Salmonella, Shigella)
Growth and colour with EMB media
It does not restrict the growth of certain bacteria but it makes them look different
We will see different colours based on how well they get broken down
What does MacConkey Agar contain?
Crystal violet, lactose, protein, and neutral red dye
What does crystal violet prevent from growing?
Gram (+)
What does crystal violet stick to?
Peptidoglycan therefore if you see growth it will be gram (-)
What growth will you see on a MacConkey Agar plate?
E. Coli: Yes
Staph Aureus: No
What growth will you see on a nutrient agar plate?
E. Coli: Yes
Staph Aureus: Yes
What type of plate type is MacConkey Agar?
Selective and differential
What happens if lactose is broken down on a plate?
There’s a colour change
What 2 things can you figure out from growth on a MacConkey Agar plate?
- If we are dealing with gram (-) or gram (+)
2. If it is something that can break down lactose
When is red dye present (MacConkey Agar)
Neutral red dye becomes red in the presence of the waste products of lactose metabolism - differential
What occurs in Alpha (group B) in blood agar?
Break down and oxidation of hemoglobin
What occurs in Beta cells (group A - streptococcus) in blood agar?
Capable of breaking down the RBCs completely, we are left with a clear area
What occurs in Gamma (group D) in blood agar?
No breakdown
What appearance do intact RBCs show in blood agar?
A nice overall red colour
What appearance do broken RBCs show in blood agar?
They become clear and get clear around the cells (differential)
What is enriched blood agar?
Blood supports additional microbes
Blood agar and sheep blood
It contains 10-20% whole sheep blood
What can you do in the hospital in terms of swabing?
There is an effective/rapid way to take swabs and get a sense of the diversity in the area and get a sense of the geno you are dealing with
How fast does E.Coli divide?
Very quickly (every 20 mins)
List the order of bacteria from fastest growing to slowest?
- E.Coli
- Staph Aureus
- Mycobacterium TB
- Mycobacterium leprae
- Treponema pallidium
How does bacterial replication occur?
Binary fission
What happens during the lag phase (Bacterial Growth Curves)
Very little change in growth, lots of metabolic activity
What happens in stationary phase (Bacterial Growth Curves)
- Metabolic rate drops, build up wastes/toxins
- Nutrients used up
What are the phases in Bacterial Growth Curves?
- Lag Phase
- Log (Exponential) Phase
- Stationary Phase
- Death Phase
What happens in the log (exponential) phase in Bacterial Growth Curves?
- Binary fission
- Exponential growth
- Very sensitive phase (ABX)
What happens in the death phase in Bacterial Growth Curves?
Cell lysis (cell ruptures)
What happens to cell division speed after the lag phase? (Bacterial Growth Curves)
It divides very quickly
When are ABX quite effective to them?
Log phase (Bacterial Growth Curves)
When are they going to compete against each other? (Bacterial Growth Curves)
Stationary Phase
Why do the cell lyse during the death phase? (Bacterial Growth Curves)
They run out of food and waste builds up
What are the steps of Virus One-step Growth Curve?
- Attachement
- Penetration
- Biosynthesis/Maturation
- Burst Time
How can you measure microbial growth? (3 words)
Direct microscope count
What is direct microscope count
Directly count the # of cells in a knwon volume using a counting chamber (hemacytometer)
What is the advantage of direct microscope count?
- Can give a good # of how many cells
- Inexpensive
- Don’t need special reagents
(Traditional way but now it can be done with equipment)
What is the disadvantage of direct microscope count?
- Tedious
- May not be able to tell if the cells are alive
How can you measure microbial growth? (2 words)
Plate count
What is plate count?
The suspension of cells is mixed with or spread on agar
What are the advantages of plate count?
Viable cells form colonies which are easily counted
What are the two plate methods
The pour plate method and the spread plate method
Which of the two plate methods are more popular?
The spread plate method
Each colony is the product of ___
A single bacterium
What is fecal coliform count?
Used to examine water samples for contamination due to human waste
How is fecal coliform count done?
A water sample is passed through a filter which is then placed on selective media (which then grow as colonies)
How can you measure microbial growth (T)
Optical Density (Turbidity)
What is optical density (turbidity)?
- Cells scatter and absorb light
- Shine light through a culture sample and measure how much is transmitted using a spectrophotometer
What will the Sepctrophotometer tell us about cells?
They will tell us how many cells are present but will not tell us if they are alive or dead
What is serial dilution?
Used if the sample is very concentrated (eg. if you were counting bacteria in a liquid culture)
- Are able to tell how many bacteria were in the original sample by working bacteria and tell the starting concentration
In what factor does dilution work (Serial Dilutions)
Each time you are reducing the # of bacteria by a factor of 10
How would you collect a sample of diseased tissue?
Surgical removal (biopsy)
How would you collect a specimen from the lung?
Either sputum dislodged by coughing or acquired via a catheter
How would you get a sample of urine?
Either sterile technique from a catheter or “clean catch” method when peeing which should be midstream
How would you get a sample from the stomach?
Intubation (inserting a tube into the stomach or an NG)
How would you get a sample of CSF?
Needle aspiration from subarachnoid space of spinal column
How would you get a sample of blood?
Needle aspiration from vein; anticoagulants are including in the specimen tube
How would you get a sample of skin?
Sterile swab brushed across the surface while not touching the neighbouring tissue
What replication process is good if you are growing bacteria in the lab
Binary fission
