Pure vs. Mixed Culture Flashcards
axenic culture
— culture that contains only one group of microorganisms which is usually obtained when microorganisms in a culture medium are all of the same species
Pure Culture
an instrument often used to transfer aliquots of culture, to
prepare serial dilutions of microorganisms, and to dispense chemical agents
Pipette
used to aseptically transfer microorganisms from broth, slant, or agar cultures to other media
Inoculating needles and loops
Pure Culture: Isolation and Characterization
Requirements:
- all apparatus and media must be previously sterilized or freed from microorganisms
- the desired bacterium must be separated from the naturally-occurring microbial populations into the sterile medium, and
- aseptic techniques which prevent the entry of contaminating microorganisms must be followed
set of specific practices and procedures performed under carefully controlled conditions with the goal of minimizing contamination
Aseptic techniques
Pure Culture Techniques
• Enrichment
• Single-cell isolation
• Membrane filtration
• Serial dilution
• Plating
isolation of specific types of microorganisms by a combination of nutrient and physical conditions
• Enrichment Culture
uses micropipette or microprobe to physically pick a single cell and transfer it on an agar medium
Single-Cell Isolation
Enrichment Culture Process
Add Sample
Incubate
Collect Sample
Colonies on Streak Plate
Pure Culture
Single Cell Isolation Process
- Target
- Suck and Hold
- Discharge at Intended Position
for samples with low population
Membrane Filter Technique
- used if the desired microorganism is present at a higher level than any other microorganism
• uses series of diuents to thin out microbial population
• Serial Dilution
The Pour Plate Method
(1.0 or 0.1 ml)
1. Inoculate Empty Plate
2. Add Melted Nutrient Agar
3. Swirl to Mix
4. Colonies grow in and on solidified medium
2 Plating Techniques
Pour plate method
Spread plate method
Spread plate method
- Inoculate Plate containing solid medium
- Spread inoculum over surface evenly
- Colonies grow only on surface of medium
3 or 4 Quadrant Methods
- Loop is Sterilized
- Loop is Inoculated
- First Set of Streaks Made
- Loop is Sterilized
- Second Set of Streaks Made
- Loop is Sterilized
- Final Set of Streaks Made
- Isolated Colonies develop after Incubation
drop/s of culture placed on a slide and overlaid with a cover glass
— observe true movement and morphology
Wet Mount Technique
Wet Mount Technique Process
- Place small drop of suspension on slide
(Edges touching will spread suspension evenly) - Gently lower coverslip
- Slide Ready for viewing
• direct microscopic count:total cell count
•standard plate count(viable cell count) and serial dilution
• most probable number(MPN)
direct method
measurement of growth
indirect methods
indirect methods
• turbidity measurement
• metabolic activity
• total weight measurement
Measurement of Growth
1. Total cell count
a. ?
b. ?
a. Direct Count
i. Counting chamber method (Petroff-Hausser chamber)
ii. Coulter counter method: automated electronic device
iii. Breed count (direct microscopic count)
b. Viable cell count – counts the number of cells or units that are capable of forming a colony
i. Pour plate
ii. Spread plate
iii. Miles and Misra (Drop method)
iv. Spiral plate method
v. Filtration
vi. Roll tube method
- for anaerobic microorganisms vii. MostProbableNumbermethod(MPN)
designed for bacteria counting and sperm analysis.
Petroff-Hausser Chamber
(Electrozone Sensing method)
Coulter Counter
measured subvolume of original sample
Aliquot
material with which the sample is diluted
Diluent
ratio of aliquot volume divided by the final volume
Dilution (D)
indicates how many times a sample has been diluted; reciprocal of dilution
Dilution factor (DF)
Dilution (D) Formula
Dilution (D) =
volume transferred/ total volume x previous D
- most probable number method
• statistical assay of cell numbers based on the theory of probability
• i.e. number of coliforms in water sample
Roll Tube Method
Measurement of Growth
2. Determination of Mass
a. ?
b. ?
a. Direct - determination of wet and dry weight
b. Indirect
i. Turbidimetric Methods
ii. McFarland standards
assay for specific biochemicals (proteins, nucleic acids) in microorganisms
Measurement by Chemical Analysis
products of microbial growth is used to estimate number of organisms or confirm their presence (i.e. acids, ATP, gases)
Metabolic Activity (Cell Products)
