Bacterial culture media Flashcards
What are the different types of Bacterial Culture Media?
- Liquid culture media- aerobic bacteria grow at the top of the tube and anaeorobic bacteria at the bottom
- Solid culture media e.g. nutrient agar for fastidious organisms; it contains bovine heart blood that becomes transparent in the presence of β-hemolytic organisms such as Streptococcus pyogenes and Staphylococcus aureus
- Selective and Non-selective e.g. blood agar
- Enriched e.g chocolate agar/ minimal media (carbon soruce, various salts and water)
- Differential e.g. MacConkey agar is differential for lactose fermentation
- Chromogenic- can visualise colonies as lots of different colours
Nutrient (non-selective) Media:
What is the typical composition?
- 0.5 % peptone
- 0.3 % beef extract/yeast extract (carbohydrate source)
- 1.5 % agar
- 0.5% NaCl (mimics bacterial cell membrane)
- Distilled water (doesn’t affect the composition of the product)
From the diagram:
Staphlococcus epidermis- 70% individuals carry this
Staphyloccocus aureus- 30% individuals carry this
Can sometimes tell the difference of what is growing
Describe the preparation of Culture Media
- 28g in 1L distilled water
- Boil to dissolve
- Sterilise by autoclaving 121ºC for 15mins
Describe the preparation of Culture Media: Agar facts
- Once melted, does not solidify until it reaches 40oC
- Cannot be degraded by most bacteria.
- Originally used as food thickener (Angelina Hesse)
Killing microbes by heating: Explain the process of autoclaving
- Close the autoclave sterilizer chamber
- Vacuum pump removes all the air from inside the device or it is forced out by pumping in steam.
- The sterilizer is pumped with high pressured steam to raise the internal temperature
- On every autoclave there is a thermometer.
- During the sterilizing process, steam is continuously entering the autoclave to kill all microorganisms.
- Once the required time of sterilization has the elapsed, the chamber will be exhausted of pressure and steam
- The door will open for cooling and drying of the contents
- Temperature in autoclave is 121°C at 15PSI (pounds of force per square inch) for 15 mins
- Autoclave tape indicated if materials are sterile:
not sterile=clear lines
sterile=dark green/black lines
From the diagram: the graph
Shallow decline at 50 degrees
Steep decline at 70 degrees
Decimal reduction times (DRT) for disinfecting testing and creating sensitivity profiles
How long it takes to drop one log order
Draw a graph representing an autoclave cycle
As the pressure increases and the temperature rises to 121°C , this is maintained for 15 mins
The door normally opens at 80°C
A complete autoclave cycle can take up to an hour
Petri dishes
Gamma irradiated to sterilise them
16ml of media and set at room temperature
turn upside down
usually store in groups of 20 in fridge
Has a shelf life of approx 1 week
What is the typical composition of enriched agar?
How is enriched agar particularly helpful?
Typical composition:
- 5 – 10% sheep or horse blood
- 0.3 % beef / yeast extract
- 0.5 % peptone
- 1.5 % agar
- 0.5% NaCl
- Determining the hemolytic capabilities of an organism.
- Some bacteria produce hemolysins that lyse red blood cells and degrade hemoglobin
- Cultivating fastidious organisms
Explain haemolysis:
Alpha, Beta and Gamma
Alpha-partial
Beta- Complete
Gamma-none
Selective culture media:
What are elective agents, selective agents and differential agents?
Elective agents:
- Encourage growth of desired organism
- e.g. High salt for Staphylococci
Selective agent:
- Desired organisms are resistant
- Bile salts for coliforms
- Antibiotics
Differential agents:
- Discriminate desired organism from others
- pH indicators
- Acid production from CHO’s
What is the typical composition of Sorbitol MacConkey Agar SMaC Agar?
Peptone 20.0 g/L
NaCl 5.0 g/L
Bile salts 1.5 g/L
Sorbitol 10.0 g/L
Crystal violet 0.001 g/L
Neutral red 0.03 g/L
Agar 15 g/L
Usually used to grow gut bacteria
Sorbitol MacConkey Agar SMaC Agar: E.Coli
- E.coli O157:H7 is a human pathogen associated with hemorrhagic colitis (certain strains of the bacterium Escherichia coli infect the large intestine and produce a toxin (Shiga toxin-SLT) that causes bloody diarrhea and other serious complications)
- Most E.coli ferments sorbitol (pink colonies)
- E.Coli 0.157 ferments lactose and does not ferment sorbital (colourless colonies) in the presence of neutral red colour indicator
What is the typical composition of Mannitol Salt Agar?
Typical composition (g/L):
Digest of casein 5.0
Enzymatic digest 5.0 of animal tissue
Beef extract 1.0
D-mannitol 10.0
NaCl 75.0
Phenol red 0.025
Agar 15.0
Oxacillin 4mg
Mannitol Salt Agar: What happens if an organism can ferment mannitol?
Will non-pathogenic staphylococci ferment mannitol?
- If an organism can ferment mannitol, an acidic by-product is formed that will cause the phenol red in the agar to turn yellow.
- Non-pathogenic staphylococci will not ferment mannitol and will apear clear on this media?
- MRSA (Methicillin-resistant Staphylococcus aureus) present when yellow colour appears
Chromogenic media- Oxoid brilliance is for the detection and enumeration of Escherichia coli and other coliforms from food and water samples
What does Rose-Gal detect?
What does X-glu detect?
Brilliance E. coli/coliform Selective Agar contains two chromogenic agents:
Rose-Gal: detects ß-galactosidase activity coliforms= pink because most organisms in the coliform group are able to ferment lactose, so will cleave the pink Rose-Gal chromogen, producing pink colonies
X-Glu: detects ß-glucuronidase activity = purple
- ß-glucuronidase is present in E. coli
- The X-Glu chromogen is targeted by this enzyme. The ability of Escherichia coli species to cleave both chromogens means that typical colonies will be purple
- Coliforms are Gram-negative nonspore formin motile or nonmotile bacteria with lactose-positive, ß -galactosidase activity, encoded by the lacZ gene
- The medium also contains sodium lauryl sulphate which acts as a selective agent, inhibiting the growth of Gram-positive organisms
- Other organisms blue/colourless
CHROMagar™ MRSA (Methicillin Resistant Staphylococcus aureus (MRSA)
- Isolation and differentiation of Methicillin Resistant Staphylococcus aureus (MRSA) including low level MRSA.
- Methicillin Resistant Staphylococcus aureus (MRSA)→ rose to mauve colonies
- Methicillin Susceptible Staphylococcus aureus(MSSA) → inhibited
- Other bacteria → blue, colourless or inhibited
Determining the viable count: serial dilution
- 9ml (of nutrient media for example) in sucessive tubes
- Add 1ml of stock solution to first tube
- Add 1ml from first tube to next tube
- First two spread plates have too many to count (TNTC)
- Last two plates dont have enough
- A good plate has 30-300 colonies
Determining the viable count: spread plates
Add 0.1ml of sample to agar and inncubate it
(colonies only grow on the top/surface)
Determining the viable count: Pour plates
Is this method more accurate than a streak plate?
Environmental labs tend to use this
- Pour plate method is usually the method of choice for counting the number of colony-forming bacteria present in a liquid specimen. In this method, fixed amount of inoculum (generally 1 ml) from a broth/sample is placed in the center of sterile Petri dish using a sterile pipette. Molten cooled agar (approx. 15mL) is then poured into the Petri dish containing the inoculum and mixed well. After the solidification of the agar, the plate is inverted and incubated at 37°C for 24-48 hours.
- Microorganisms will grow both on the surface and within the medium. Colonies that grow within the medium generally are small in size and may be confluent; the few that grow on the agar surface are of the same size and appearance as those on a streak plate.
- Colonies grow on the top (aerobic) and the center of the media (anaerobic)
- The pour plate method of counting bacteria is more precise than the streak plate method, but, on the average, it will give a lower count as heat sensitive microorganisms may die when they come contact with hot, molten agar medium.
Give an example of how to calculate the TVC?
- 60 cfu on a 10-4dilution with 0.1ml spread
- 60 x 10 = 600 cfu in 10-4 in 1ml
- 600 x 104 = 6000000 cfu per ml
- 6.0 x 106 cfu / ml
Wire innoculating loops
Streak plates
Other Selective Agents
- Incubation conditions:
37ºC
44ºC for E.coli
30ºC for ACC
- Atmosphere:
Aerobic
Anaerobic
Microaerobic
Describing Bacterial Colonies
- Size
- Shape
- Texture
- Pigmentation
API-Biochemical profiling
- The API (Analytical profile index) system is a classification of bacteria based on experiments, allowing fast identification. This system is developed for quick identification of clinically relevant bacteria.
- Because of this, only known bacteria can be identified.
- combines some conventional tests and allows the identification of a limited number of bacteria
- The test systems are stored in 20 small reaction tubes, which include the substrates (sugars)
- Take sample and make 0.5 McFarlan standand
- Add to each of 20 wells and leave to incubate overnight
One of the API systems is specific for differentiating between members of the Gram negative bacterial Family Enterobacteriaceae and is called API-20E. The other API system is specific for Gram positive bacteria, including Staphylococcus species, Micrococcus, species, and related organisms, and is called API-Staph
Scoring an API strip
- API test strips consists of wells containing dehydrated substrates to detect enzymatic activity, usually related to fermentation of carbohydrate or catabolism of proteins or amino acids by the inoculated organisms. A bacterial suspension is used to rehydrate each of the wells and the strips are incubated. During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents
- Add the numbers depending if there is a positive or negative result to get a unique identificaiton number
- All positive and negative test results are compiled to obtain a profile number, which is then compared with profile numbers in a commercial codebook (or online) to determine the identification of the bacterial species
Vitek 2
- Detects bacterial growth and metabolic changes in the microwells of the thin plastic cards by using a fluorescence based technology.
- It has different microwell cards that contain antibiotics or biochemical substrates
- An integrated modular system that consists of a filling-sealer unit, a reader-incubator, a computer control module, a data terminal and a read incubator
MALDI-TOF- Matrix Assisted Laser Desorption/Ionization
- Introduced into a high vacuum environment.
- The sample is ionized with a precise laser burst, releasing a “cloud” of proteins.
- These proteins are accelerated using an electric charge, and the time of flight is recorded; lighter proteins travel faster, heavier proteins travel slower.
- The proteins are detected with a sensor
- This creates a spectrum representing the protein makeup of each sample.
- By comparing the spectrum from a particular sample against a large database of spectra from precisely characterized bacteria and fungi, identifications can be made at the species, genus and family level.
What is an infectious dose?
Minimum number of bacterial cells required to cause infection in humans
would you report 0 cfu/ml?
No
You would report <1cfu/ml
