Microbial Culturing Flashcards
What should you consider when trying to collect specimens?
-Containers should be sterile with proper lids
- Specimens may need to be stored or shipped at specific temps
- details on labels are important
- other special instructs may apply
Ie) urine sample taken mid-stream
How fast does Escherichia coli double in?
17-20 minutes
What are cultures used for?
Identification/characterization of a microorganism, determining is abundance, fermentation products and scientific research
What is a mesophile?
Most organisms fit into this category. They grow between 20-40 degrees
What is a thermophile?
Likes heat: grows between 40-80 degrees
Listeria monocytogens grows best at _____________, but also grows well at _________________. It is known as a __________________, because it can grow in the _____________
30 degrees, colder 0 degrees, psychotrophic. Cold
What bacteria causes listeriosis
Listeria monocytogens
What is the leading cause of death among food borne bacteria pathogens and why?
Listeria monocytogens. It can grow in the cold and at 30 degrees.
How does listeria monocytogens infect?
Secretes invasion that enables the bacteria to penetrate the host cell and move to other tissues.
Define what an acidophiles, neutrophils, alkaliphile is.
Acidophiles: like acid: 1-5.5
Neutrophils: neutral: 5.5-8.5
Alkalphile: basic, 7.5-11.5
What pH do most fungi prefer?
Acidic ph ~5-6
What pH do most bacteria prefer?
~6-8
Give an example of a type of bacteria that can survive in your stomach.
Helicobacter pylori: associated with gastric ulcers and cancers
What is osmotic pressure (how salty)
Determines whether water will be trying to move or out of cell: can lead to dehydration or to cell exploding due to to high of water inside cell
What is an isotonic solution?
No water movement. Solutes in side and outside the cell are equal
What is a hypertonic solution?
There is more salt/solutes outside the cell than inside and water exists the cell
Give an example of a type of bacteria that likes a salty or hypertonic solution and where it lives?
Straphlococcus epidermis: lives on your skin which is salty
What is a halotolerant organism?
(Staphylococci) can tolerate salty envrionments
What are the most common chemical requirements?
Crabon: for organic molecules, energy source
N: aa,& NT
S: aa, thiamine, biotin,
P: DNA,RNA, ATP, phospholipid membrane
Organic factors: somthing that the organism can not produce on its own. Ie) vitamins
Trace elements: inorganic elements ie) Fe
O2: obligate, facultative: aerobes/anaerobes
What does reducing media do?
Contains chemicals that react with and remove 02 from the media
How to you obtain a single distinct colony in a culture in a lab
The easiest technique is streaking
What is a colony morphology?
Microbes can be defined on media (liquid or solid) by the colony colour, texture, shiny, translucent…
What is the purpose of streaking?
To isolate a pure strain from a single species of microorganism.
How do you culture virus?
You must first culture the host cell than can use the host cell to culture the virus.
How do you culture bacteriophages?
They are grown on a bacterial lawn. The spots that are clear “plaques” are where the virus has grown.
Where are animal virus cultured?
-Embryonated eggs
Ie) influenza is modified to grow in eggs, allows to culture for vaccine.
-Tissue/cell culture ie) polio virus
-living animals ie) HIV in modified mice., Nora virus (need human volunteers causes explosive Diaherria and vomiting)
Why are procedures performed under sterile conditions?
1) To keep the culture pure and uncontaminated.
2) protect workers/lab
What are the different types of growth media?
- General/complex
- chemically defined
- enriched
- selective
- differential
Explain what a general/complex media is:
-non-selective
-encourages the growth of a range of microbes
Ie) Nutrient Agar, TSA (trytic Soy Agar)
Explain what a chemically defined growth media is:
The exact chemical composition is precisely known. ->often used to study genetic mutants
What is an enriched media?
Non-selective-> contains additional factors needed to grow microbes with particular needs.
Ie) chocolate agar: has Lysed (typically sheep) blood so that the hemoglobin is exposed.
What is a selective media?
Only supports the growth of specific microbes.
Often used to isolate a specific species.
Ie) pH of media to low for fungi to grow but not bacteria.
Ie)an antibiotic is added so that Pseudomonas aeruginosa can grow but E.coli can’t
What is differential media?
The agar will allow for different appeared due to the characteristics of the media (turn different colours)
Ie)EMB (Eosin Methylene Blue) agar: provides a colour indicator distinguishing between organisms that ferment lactose (E.coli) and those that don’t ie) Samonella
MacConkey Agar is used for what (Very important to know this!)
-isolate G (-)
-contains crystal violet stain (kills G (+), protein and neutral red dye. (Makes it selective)
-Differential: b/c the neutral red dye becomes red in the presence of the waste products of lactose metabolism.
The neutral red dye interacts with how acidic/basic it becomes.
Ie) E.coli = red
Salmonella = yellow
What is Blood Agar used for?
To isolate pathogens with complex nutritional requirements and to detect hemolytic activity.
How much blood does blood agar contain?
5-10% WHOLE sheep blood
Why does blood agar need to be enriched?
It is enriched with blood to support the growth of additional microbes.
What makes Blood Agar differential?
A clear ring is seen round colonies of bacteria which are cable of hemolysis (lysine red blood cell)
->good for streptococcus
What are the different types of hemolysis:
Alpha- hemolysis
Beta- hemolysis
Gamma- hemolysis
What is alpha-hemolysis and give an example.
Partial hemolysis-> appears greenish on agar.
Ie) Streptococcus pneumoniae
What is Beta-hemolysis and give examples (2)
Complete hemoloysis-> appears clear Group A (GAS): Streptococcus progenes (strep throat) Group B (GBS): Streptococcus agalactiae (infectious to new borns)
What is Gamma hemolysis and give an example.
No clearing (no-colour) (formily Group D): Enterococcus faecalis
Besides growing conditons how else can you identify bacteria?
By Bacterial enzymes and by-products of metabolic processes.
What is the Catalase test?
The catalase test is used to differentiate staphylococci (catalase +) from streptococci (catalase -)
What does the catalase test do?
Bubbles of 02 gas are produced when peroxide is added to a smear of culture (catalase positive)
Catalse: H202 (peroxide)-> H2O +O2
What is the coagulate test do?
Coagulase is an enzyme produced by Staphylococcus aureus that coverts soluble fibrinogen (human protein) in plasma to (insoluble) fibrin.
How do you perform a coagulase test?
A drop of rabbit plasma is added to a smear of culture. Clumping of the plasma indicates coagulase-positive.
What is the urease test do?
Proteus sp. produces urease. The specimen is placed in media containing urea and pH indicator. As urea is broken down it produces ammonia and raises the pH
What is a rapid identification system?
Various systems are available…. typically allows multiple test to help identify an organism quickly.
What is antimicrobial sensitivity testing:
Sometimes need to both identify both the organism and what antibiotic therapy may be effective.
What do microbes grow in naturally as complex communities?
Biofilm
What method to you use to get a pure culture?
Streaking
How to you culture a virus cell?
Using host cells
What are liquid & solid media, and why are they used?
Liquid: broth-> some things won’t form colonies. Ie) protist
Solid: agar: allows to isolate indivuial bacteria to grow pure colonies.
Why must media, flask/tubes and instruments be sterilized before used for growing microbes?
To make sure there is no cross-contamination
What are bacterial lawns used for?
To grow phages
What types of chemical and physical requirements must be considered for culturing microbes?
Temp, pH, nutrient requirements, presence of 02
How would you describe MacConkey agar and blood agar?
MacConkey agar:
Selective: uses crytal violet to kill G (+)
Differential: uses neutral red dye to distinguish the metabolism of lactose
E-.coli = red
Samonella = yellow
Blood agar:
Enriched: whole blood was needed.
Differential: clear ring/colour based on hemolysis of cell
Alpha: partial/green
Beta: complete/ clear
Gamma: non-hemolytic= no colour.
