LAB EXAM 2 Flashcards
Culture Media Preparation (6)
- Weighing of dehydrated media or media ingredients.
- Dissolving of components in the solvent.
- Checking or adjustment of pH (optional).
- Dispensing into glassware or containers.
- Capping or plugging with cotton.
- Sterilization (usually at 15 psi, 15 minutes).
other name of pure culture
axenic culture
a 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 or axenic culture
Instruments used to maintain pure culture:
- Pipette
- Inoculating Loop
an instrument often used to transfer aliquots of culture; to prepare serial dilutions of microorganisms; and to dispense chemical agents.
Pipette
are used to aseptically transfer microorganisms from broths, slants, or agar cultures to other media.
Inoculating Loop
Requirements to get pure culture (3)
- 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.
- Aseptic techniques which prevent the entry of contaminating microorganisms must be followed.
a set of specific practices and procedures performed under carefully controlled conditions with the goal of minimizing contamination.
Aseptic Techniques
Pure Culture Techniques: (5)
- Enrichment
- Single-Cell Isolation
- Membrane Filtration
- Serial Dilution
- Plating
isolation of specific types of microorganisms by a combination of nutrient and physical conditions.
Enrichment
uses a 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
Pure culture technique for samples with low population.
Membrane Filtration
used if the desired microorganism is present at a higher level than any other microorganism; uses a series of diluents to thin out microbial population.
Serial Dilution
Plating (3)
Pour Plate Culture
Spread Plate Culture
Streak Plate Culture
drop/s of culture placed on a slide and overlaid with a cover glass; observes true movement and morphology.
Wet Mount Technique
Measurement of Growth is measured by following changes in the _______or _______ of cells.
number or weight
Direct Methods for measurement of growth (3)
- direct microscopic count: total cell count;
- standard plate count and serial dilution
- most probable number (MPN)
indirect methods of measurement of growth (3)
- turbidity
- metabolic activity
- total weight measurement
Two types of total cell count
Direct count
Viable cell count
examples of direct count method (3)
- Counting Chamber Method (Petroff-Hausser Chamber)
- Coulter Counter Method (Automated Electronic Device)
- Breed Count (Direct Microscopic Count)
Viable Cell Count (7)
- Pour Plate
- Spread Plate
- Miles and Misra (Drop Method)
- Spiral Plate Method
- Filtration
- Roll Tube Method
- Most Probably Number Method (MPN).
What viable cell count method is for anaerobic organism?
Roll tube method
counts the number of cells or units that are capable of forming a colony
Viable cell count
- 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.
Coulter Counter (Electrozone Sensing Method)
What kind of total cell count method (direct method) is this?
- 0.01 mL of milk sample
- Evenly spread out on 1 sq. cm area on the glass slide; air dried
- Cover the area with xylol for 1 minute; drain; air dry
- Flood with alcohol for 1 minute; drain, air dry
- Cover with methylene blue for 3-5 minutes; wash with water
- Drain and dry without blotting, examine under OIO
Breed count
measured subvolume of original sample
Aliquot
material with which the sample is diluted
Diluent
ratio of aliquot volume divided by the final volume
Dilution
Dilution Equation
Dilution (D) =
(volume transferred/total vol) x previous D
indicates how many times a sample has been diluted; reciprocal of dilution
Dilution Factor (DF):
is a step-wise series of dilutions, where the dilution factor stays the same for each step
serial dilution
num of colonies on plate x reciprocal of dilution of sample =
num of bacteria /ml
if 32 colonies are on plate of 1/10,000 dilution, then the count is?
32 x 10,000 = 32,000
Valid Counts of
bacteria:
fungi:
bacteria: 25-250 per plate
fungi: 10-150 per plate
if one dilution give valid counts, what CFU/ml will be used?
CFU/ml = (ave. num of colonies x DF) / volume plated
if two dilutions give valid counts, what CFU/ml will be used?
CFU/ml = (summation of num of colonies / {[(n1x1) + (n2x0.1)] x d}) / volume plated
where n1 = num of plates in lower dilution
n2 = num of plates in next higher dilution
d = lower dilution
method used to save money? kay gina divide ang culture media to grow microorganism
Miles and Misra method which is a drop plate method
method na pina spiral ang growth
Spiral Plate method
spiral method formula
n = N / V x D
where
n : Bacterial concentration at CFU/mL
N : Number of counted colonies
D : Dilution factor of the sample
V : Deposited volume in the counted sector in
μL (Cf. Volumes Constants)
- 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
Determination of Mass (2)
Direct and indirect
determination of wet and dry weight
direct method of determination of mass
indirect method of determination of mass (2)
i. Turbidimetric Methods
ii. McFarland standards
assay for specific biochemicals (proteins, nucleic acids) in microorganisms
Measurement by Chemical Analysis
in tubidimetry,
if more cells present, _____ light scattered, ____ light transmitted
more, less
detects amount of unscattered light
spectrophotometer
in turbidimetry,
is proportional to the number
of cells but inversely proportional to the
measurement that reaches the light detector
optical density
standards that are used for adjusting densities of bacterial suspensions.
McFarland Standards
is used to estimate number of organisms or confirm their presence (i.e. acids, ATP, gases)
products of microbial growth
Gram Staining Method is named after Danish bacteriologist who originally devised it in 1882 (published in 1884)
Hans Christian Gram
reflects fundamental differences in the biochemical and structural properties of bacteria.
Gram reaction
Gram Staining Method (4)
Crystal Violet – Primary Stain
Iodine – Mordant
95% Ethanol – Decolorizer
Safranin – Counterstain
Primary Stain
Crystal Violet
Mordant
Iodine
Decolorizer
95% Ethanol
Counterstain
Safranin
0.5 Mcfarland Turbidity Std. in CFU/ml
1.5 x 10^8 CFU/ml
- Gram negative bacteria (pink; polychromatic; complex medium)
- a selective and differential medium used to isolate fecal coliforms. Eosin Y and methylene blue are pH indicator dyes which are combined to form a dark purple precipitate at low pH.
- serves to inhibit the growth of most Gram-negative organisms (e.g., E. coli).
Eosin Methylene Blue (EMB) Agar
- Gram positive bacteria (violet).
- a commonly used selective and differential growth medium in microbiology.
- encourages the growth of a group of certain bacteria while inhibiting the growth of others.
Mannitol Salt Agar (MSA)
color of E.coli in EMB
green (pero purp ako :<)
color of STAPHYLOCOCCUS AUREUS in MSA
yellow/white
is a loose agar that allows for better diffusion of the antibiotics than most other media.
MHA (Mueller Hinton Agar)
direct microscopic count
total cell count
standard plate count
viable cell count
Example of Counting Chamber Method
Petroff-Hausser Chamber
an automated electronic device that directly counts the total cell count
coulter counter method
example of coulter counter method
- particles in dilute electrolyte suspension and uses voltage pulse to record the particles that flows through the orifice
electrozone sensing method
example of direct microscopic count that uses milk
breed count
in _______ plate method, colonies grow in and on solidified medium while in _______ plate method, colonies grow only on surface.
pour, spread
