Pure culture: Isolation and characterization Flashcards
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
relating to or denoting a culture that is free from living organisms other than the species required.
Axenic culture
Instruments used to maintain pure culture (2)
Pipette
Inoculating needles and loops
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 (3)
All apparatus must be sterilized
Desired bacterium must be separated from NATURALLY-OCCURING microbial population
Aseptic techniques used
set of specific practices and procedures performed under carefully controlled conditions with the goal of minimizing contamination
Aseptic techniques
Pure Culture Techniques (5)
Enrichment Culture
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
Single-cell isolation steps (3)
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 diluents to thin out microbial population
Serial Dilution
Plating Techniques (3)
Streak plate method
Pour plate culture
Spread plate culture
drop/s of culture placed on a slide and overlaid with a cover glass
observe true movement and morphology
Wet Mount Technique
Pour plate method steps (4)
Inoculate empty plate (1.0 or 0.1 ml)
Add melted agar
Swirl to mix
Colonies grow in and on solidified medium
Spread plate method (3)
Inoculate plate containing solid medium (0.1 ml)
Spread inoculum over surface evenly
Colonies grow only on surface medium
3 or 4 quadrant methods (8)
Loop sterilized
Loop INOCULATED
First set of streaks
loop sterilized
Second set of streaks
Loop sterilized
Final set of streaks made
Isolated colonies develop
measured by following changes in the number of cells or weight of cells
Measurement of Growth
Direct methods of Measurement of growth (3)
Direct Microscopic count: Total cell count
Standard plate count (viable cell count) and serial dilution
Most probable number (MPN)
Indirect Methods of Measurement of growth (3)
turbidity measurement
metabolic activity
total weight measurement
Total cell count: Direct count (3)
Counting chamber method (Petroff-Hausser chamber)
Coulter counter method: automated electronic device
Breed count (direct microscopic count)
counts the number of cells or units that are capable of forming a colony
Viable cell count
Total cell count: Viable cell count (7)
Pour plate
Spread plate
Miles and Misra (Drop method)
Spiral plate method
Filtration
Roll tube method - for anaerobic microorganisms
Most Probable Number method (MPN)
Slide used in Counting chamber method
Haemocytometer slide
Counting chamber method (4)
Bacterial suspension added and fills shallow volume over squares by capillary action
Volume calculated (depth x area)
Microscopic count: all cells are counted and numbers are averaged
Volume of fluid over the large square is 1/1,250,000 of a milliliter
SO, if ther are 14 cells shown then there are 14 x 1,250,000 cells in a milliliter
Also known as the ___
Particles are held in suspension in a DILUTE ELECTROLYTE which is drawn through the ORIFICE with a VOLTAGE applied across it
VOLTAGE PULSE is recorded as particles flow through the orifice
Amplitude of pulse can be related to volume of particle
Counter Counter method
Electrozone Sensing method
Breed count used in milk steps (6)
0.01 mL milk sample
Spread out on 1 sq.cm area on glass slide, air dried
Cover area with XYLOL for 1 min, air dry
Flood with ALCOHOL for 1 min, drain, air dry
Cover with METHYLENE BLUE for 3-5 minutes; wash with water
Drain and dry without blotting, examine under oio
measured subvolume of original sample
material with which the sample is diluted
Aliquot
Diluent
Dilution (D): ratio of aliquot volume divided by the final volume
Dilution (D) =
(volume transferred / total volume) x previous D
Drops applied
Applied in circular motion through a machine
Surface of tube covered with GAS IMPERMEABLE STOPPER has agar
Miles and Misra method
Spiral Plate Method
Roll Tube Method
Filtration method steps (5)
Membrane filter possessing millipores small enough to trap bacteria
Water sample filtered through the filter to trap bacteria on the filter
Membrane filter removed and placed in a petriplate filled with appropriate medium
Petriplate incubated for 24 hours
Typical colonies develop
Measure of growth Other types (2)
Determination of Mass
Measurement by Chemical Analysis
Determination of mass (2) (2)
Direct - wet and dry weight
Indirect - Turbidimetric methods
- McFarland standards
assay for specific biochemicals (proteins, nucleic acids) in microorganisms
Measurement by Chemical Analysis
- more cells present, more light scattered, less
light transmitted - ____ – detects amount of
unscattered light - ____ is proportional to the number
of cells but inversely proportional to the
measurement that reaches the light detector
Turbidimetry
Spectrophotometry
optical density
Used as basis of turbidity
McFarland Standard
McFarland composed of?
Anhydrous BaCl2
H2SO4
products of microbial growth is used to estimate number of organisms or confirm their presence
Metabolic Activity (cell products)
