oleh : Iman Santoso

Lab. Mikrobiologi – Departemen Biologi, Kampus UI Depok.

Enumeration of microorganisms is especially important in dairy microbiology, food microbiology, and water microbiology.

Bacterial Enumeration

The measurement of the number of bacterial cells per milliliter, gram, or cubic meter of a sample , depends on the nature of the sample.

Standard Plate Count is one of enumeration method. It is done by pipetting a fixed volume (usually 0,1 ml) of sample, plating aliquots onto an appropriate culture medium, spread across the plate, then incubating the plates under proper conditions. After incubation, colonies are formed. The colonies are counted and referred as colony forming units (CFU)

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For accurate determination of

the total number of viable cells, it is critical that each colony comes from only one cell, so chains and clumps of cells must be broken apart. However, since one is never sure that all such groups have been broken apart, the total number of viable cells is usually reported as colony-forming units (CFUs) rather than cell numbers.

Bigger

Smaller

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When using standard size Petri dishes (100 mm), a countable plate would be one with between 30 and 300 CFUs or between 20 and 200 colonies for 50 mm plates are counted which is usually resulted from 0.1 ml sample.

But usually high numbers of bacteria are often present in samples. Some samples may contai 108 cells/ml. At this concentration, a 0.1 ml volume plated onto a nutrient agar plate could yield 10,000,000 colonies, which is mean impossible to calculate accurately.

To overcome this problem, serial dilutions of the sample prior to plating is necessary, so when a small volume of the diluted sample is plated, the colonies are countable. Ex.: sample containing 108

cells/ml will require a dilution of 10-6 to achieve a countable number of 100 CFU.

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A serial dilution is any dilution where the concentration decreases by the same quantity in each successive step.

EXAMPLE: What is the dilution factor if you add 0.1 mL aliquot of a specimen to 9.9 mL of diluent?The dilution factor = The aliquot volume : Total final volume = 0.1 ml : (0.1 ml + 9.9 ml) = 0.1 ml : 10 ml = 1 : 100

Since the number of cells present in a sample is unknown, it is necessary to make several dilutions so that some dilution will contain a countable number of cells when plated. As a general rule, the more turbid the suspension, the greater the number of organisms in the suspension.

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The highest dilutions will produce the lowest number of CFUs and the The highest dilutions will produce the lowest number of CFUs and the lowest dilutions will produce the highest number of CFUslowest dilutions will produce the highest number of CFUs

Higher Dilution

Lowest Number

This method of enumeration is relatively easy to perform and is much more sensitive than turbidimetric measurement. A major disadvantage, however, is the time necessary for dilutions, platings and incubations,as well as the time needed for media preparation.

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SERIAL DILUTIONS

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SERIAL DILUTIONS

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Total Plate Count (TPC)

• Approximation of total microbial load in the food

• Method detects aerobic, mesophilic microorganisms

• Microbial populations are expressed as colony forming units (CFU) per gram or milliliter

TPC gives an ESTIMATE- Why?

• Obligate anaerobes not counted• Clumps of cells can produce a single colony leading to overestimation• May be affected by environmental constraints (incubation temperature, pH)

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Plating on AgarPlating on Agar

• Prepare 2 plates per dilution

• Add 0.1 ml of sample to the agar plates (label plates first)

• Spread-plate the inoculum with a hockey stick

• Invert the plates

• Incubate sample plates: 35°C for 48 h

• Incubate sample plates: 30°C for 4 days (observe them after 48 h without opening the plates!)

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TPC CALCULATIONTPC CALCULATION

To determine the concentration of bacteria in the original sample, the colonies are counted, the count is divided by the volume plated, and then multiplied by the reciprocal of the total dilution factor:

CFU/ml =Number of CFU X (1/Volume Plated) X (1/dilution factors)

Suppose that a 0.1 ml sample, which is represent from a (1/1,000,000) dilution of the original sample, was plated and after incubation, the plate had 50 colonies.

Then the original sample must contain concentration of bacteria : = 50 x (1 / 0.1) x (1 / 106) = 50 x ( 10 ) x (106) = 500,000,000 cfu/ml (5.108 cfu/ml)

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To increase the accuracy of the results, it is best to plate the samples in duplicate or triplicate and then determine the average count. Major sources of error include inaccurate volumes of diluent and/or inaccurate transfer of volumes. Bacterial cells that adhere to each other give rise to a single colony and produce low estimations of bacterial numbers.

Pour Plate method: The most commonly used method for enumeration of bacteria in

a wide variety of samples including milk, food, meat, soil etc.

Pour plate methods yield a count of only the living cells in the sample and thus are a viable count.

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PERHITUNGAN PERHITUNGAN ANGKA LEMPENG TOTAL (ALT)ANGKA LEMPENG TOTAL (ALT)

PROGRAM STUDI MIKROBIOLOGISEKOLAH ILMU DAN TEKNOLOGI HAYATI

INSTITUT TEKNOLOGI BANDUNG2009

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ANGKA LEMPENG TOTAL (ALT)ANGKA LEMPENG TOTAL (ALT)

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PERHITUNGAN KOLONI BAKTERI (1)PERHITUNGAN KOLONI BAKTERI (1)

Dipilih cawan petri dari satu pengenceran yang menunjukkan jumlah koloni antara 25-250 koloni

Jumlah koloni rata-rata Jumlah kedua cawan dikalikan dengan faktor pengencerannya

Maka Angka Lempeng Total adalah :

150 + 250 x 102 = 200 x 102

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PERHITUNGAN KOLONI BAKTERI (2)PERHITUNGAN KOLONI BAKTERI (2)

Bila salah satu dari cawan petri menunjukkan jumlah koloni ≤25 atau ≥250 dihitung jumlah rata-rata koloni, kemudian dikalikan dengan faktor pengencerannya

Maka Angka Lempeng Total adalah :

250 + 200 x 102 = 225 x 102

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PERHITUNGAN KOLONI BAKTERI (3)PERHITUNGAN KOLONI BAKTERI (3)

Bila cawan-cawan dari dua tingkat pengenceran yang berurutan menunjukkan jumlah koloni antara 25-250 dihitung jumlah koloni dari masing-masing tingkat pengenceran, dikalikan dengan faktor pengencerannya

Maka Angka Lempeng Total adalah :220 + 280 x 102 = 250 x 102

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PERHITUNGAN KOLONI BAKTERI (4)

Bila hasil perhitungan pada tingkat pengenceran yang lebih tinggi diperoleh jumlah koloni rata-rata ≥2 kali jumlah koloni rata-rata pengenceran dibawahnya, maka dipilih tingkat pengenceran yang lebih rendah

Maka Angka Lempeng Total adalah 140x102

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PERHITUNGAN KOLONI BAKTERI (5)PERHITUNGAN KOLONI BAKTERI (5)

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PERHITUNGAN KOLONI BAKTERI (6)PERHITUNGAN KOLONI BAKTERI (6)

Bila tidak satupun koloni tumbuh dalam cawan, maka Angka Lempeng Total dinyatakan sebagai <1 dikalikan faktor pengenceran terendah.

Maka Angka Lempeng Total adalah <1x 102

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PERHITUNGAN KOLONI BAKTERI (7)PERHITUNGAN KOLONI BAKTERI (7) Jika seluruh cawan menunjukkan jumlah koloni ≥250,

dipilih cawan dari tingkat pengenceran tertinggi kemudian dibagi menjadi beberapa sektor (2,4, atau 8) dan dihitung jumlah koloni dari satu sektor

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Angka Lempeng Total jumlah koloni dikalikan dengan jumlah sektor, kemudian dihitung rata-rata dari kedua cawan dan dikalikan dengan faktor pengenceran

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PERHITUNGAN KOLONI BAKTERI (8)PERHITUNGAN KOLONI BAKTERI (8)Jika jumlah koloni rata-rata dari 1/8 bagian cawan ≥200, maka

Angka Lempeng Total dinyatakan ≥ 200x8 dikalikan faktor pengenceran

Maka Angka Lempeng Total adalah ≥ 200x8 102

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PENCATATAN HASIL ALTPENCATATAN HASIL ALT

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