Methods for the Microbiological Examination
MIKROBIOLOGI DASAR
Methods for the
Microbiological Examination
By :
Syarifah Hikmah Julinda, S.Pi, M.Sc
Basic group of microbiological examination
Number Cell Count
Mass Count
Total Cell Count
Viable Cell Count
DEPEND ON
The type of information required
The number and types organisms presents
The physical nature of the sample
Enumeration of Microbes
Total cell count
Direct microscopic count of microbial cells
Plate count method
Most Probable Number (MPN) method
Direct cell mass count
Indirect cell mass count
Total cell count - Direct microscopic Count
Microscopic counts of microbial cells may be counted directly by observing the total number cells either alive or died microbes in a counting chamber
This is done by placing a suspension in a counting chamber
The glass slides with measured grid is prepared with a coverslip
When a coverslip is placed over the suspension, this fixed volume of sample over each square in the grid
The mean number of microbes lying over each square of grid is calculated by direct counting
The equipments are Petroff-Hausser, Haemacytometer,
Bacteria Counter, Colony Counter, etc
Petroff-Hausser Method
The method is used to calculate the total cell of microbes in the small sample
The glass slides is in square form with fixed volume
In 1 mm 2 of glass slides fill with 25 square (size of 0,04 mm 2 ).
Each square consists of 16 small square. The high between glass slides and cover slips is 0,02 mm. Total of cell number in ml sample can be calculated as follows : number cell in the large square x 25 square x 1/0,02 x 10 3
Ex. There are 12 cells, so the total number per ml per sample is
12 x 1,25 x 10 6 = 1,5 x 10 7 cell/ml
Quick count
Cheap
Easy to count
Plus Minus
There is no different cells calculation for live and died microbes
The cell that own small size is difficult to count
It need high accuracy
The sample need to be free from debris and food extraction
Breed method
Bread method used to assess the quality of milk to give an indication of number of the bacteria and leucocytes in milk.
10 microlitre of milk is taken up by a Breed pipette and delivered onto a 1 cm2 a microscope slide,
The milk is spread over the squared with a straight inoculating wire
The film is air dried and stained with Newman’s stain
The number of bacteria and leucocytes in ten randomly selected fields are counted
Since the are 3000 high power fields per cm2 and 10 microlitre of milk was spread over this area, the number of bacteria and leucocytes present in the original sample can be calculated by multiplying the average count per high power field by 3 x 10 5 per ml
Cheap technique
Plus
Rapidly carried out
Yield valuable information about milk sample
Minus
There is no different cells calculation for live and died microbes
The cell that own small size is difficult to count
It need high accuracy
Total cell count – Total Plate Count/Standard Plate
Count/Aerobic Plate count
It’s a viable count
The aerobic plate count (APC) is used as an indicator of bacterial populations on a sample. It is also called the aerobic colony count, standard plate count, mesophilic count or total plate count.
The test is based on an assumption that each cell will form a visible colony when mixed with agar containing the appropriate nutrients.
Clumps of cell => 1 colony => CFU (colony-forming-units)
The number of colonies depends on:
1.
Inoculum size
2.
3.
4.
Inoculum conditions
Selecting Culture medium
Length and temperature of incubation
Pour Plate Method
This method involves the use of larger samples from each dilution
(1ml or 0,1 ml)
The samples are mixed with molten, cooled batched (47-50 0 C) of growth medium (15-20 ml). Each dilution-growth medium mixture is the poured into its own sterile petri disk
Plate are incubated as usual
The number of colonies both on and within the solid medium may be counted as for the pour-plate technique
The calculation = the total number of colonies x 1/dilution factors
Video pour plate method
Spread plate technique
The initial inoculum is subjected to a serial dilution to a point where no bacteria are expected to be present in the diluent
A small sample is the removed from each dilution tube and placed on the surface of an appropriate solid growth media
The inoculum is spread over the surface of plate using a sterile spreader
The cultures are incubated
Video spread plate technique
The Differentness
Spread Plate Pour Plate
It applied when the microbe to be counted grows best on the surface the culture medium
It used when opaque medium agar is required to support the growth of the organisms
Its important when counting highly motile bacteria
This method is not suitable for heat-sensitive microbes
Obligate aerobes may exhibit a slight reduction
Typical colonial morphilogies are different from surface culture
Calculation
Spread plates:
30-200 colonies
Pour plates:
30-300 colonies
1
2
No Dilution Total colony in a petri disk The total count microbes per
1 2 ml
10 -4
10 -5
10 -4
10 -5
350
24
300
62
280
25
Spreader
50
2.800.000
4.300.000
3
4
10 -4
10 -5
10 -4
10 -5
315
25
250
70
Spreader
20
270
80
3.150.000
2.600.000
Plus
It Calculate alive microbes only
Some microbes can be calculate together
It can use to isolate and identify microbes
Minus
The results is not represent the real number of microbes. It calculate only the number of microbes that grow on the specific medium under particular growing conditions.
Its needs preparations and length of incubation to sea the colonies
It is difficult to distinguish between feed particles and bacteria.
Total cell count-MPN
The "most probable number" (MPN) method is a method to estimate the concentration of viable microorganisms in as ample by means of replicate liquid broth growth in ten-fold dilutions
The method is particularly useful with samples that contain particulate material that interferes with plate count enumeration methods.
The basic concept to the MPN method is the nutrient broth will support growth of organisms and turn turbid.
The method
The sample to be tested is prepared in 10-fold dilution series
1mL samples of each dilution are inoculated into triplicate broth culture tubes for incubation.
Following incubation, all tubes are examined for turbidity and the pattern of growth in the tubes is scored against a table of such values
The MPN table normally only presents results for three dilutions in sequence
Direct cell mass count-
Turbidimetric counting
Microorganisms have ability to scatter light and to appear turbid
The turbidity of a microbial suspension is proportional to the number of cell present
The method for emasureing light scattering
The nephelometer : Measure the amount of light scattered directly by using a photoelectric detector placed at right angles to the light source
The spectrophotometers : measure the dispersal light by placing the sample directly in line with both the light source ad the photoelectric detector. This measure the light lost from a culture after it has passed the microbial suspension. The reading are expressed as absorption or optical densities (OD).
The wavelength used is 600 nm
The absorbance (OD) is linear to the cell number
Mcfarland standard tubes
Direct Method
Filtration
Wet and dry weight counting
Microbial growth resuts in the production of novel biomass
Suspensions of microbes may be harvested by centrifugation
The wight of cells determined
The cell mass is made by measuring of the wet weight of the deposit from suspension
To determine the value, the deposite is placed into hot oven to eleminate waters and estimate as the dry wet
Indirect cell mass count
Cell components analysis (protein, ATP, ADP)
Catabolisms product analysis (primary metabolite, secondary metabolite)
Nutrient consumption analysis (C, N)
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