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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Smaller

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.

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Bigger
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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.

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 lowest dilutions will produce the highest number of CFUs
Higher Dilution

sample .... ..... ... ..... ...... .... .... ..... ... ... .. ...

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

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 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: 35C for 48 h Incubate sample plates: 30C for 4 days (observe them after 48 h without opening the plates!)

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TPC 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 ANGKA LEMPENG TOTAL (ALT)

PROGRAM STUDI MIKROBIOLOGI SEKOLAH ILMU DAN TEKNOLOGI HAYATI INSTITUT TEKNOLOGI BANDUNG 2009

Angka Lempeng Total (ALT)

Total Plate Count (TPC)

Standard Plate Count (SPC)

Aerobic Microbial Count (AMC)

Aerobic Plate Count (APC)

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ANGKA LEMPENG TOTAL (ALT)

Jumlah mikroba aerob mesofil dalam suatu produk
-Umumnya tidak selalu terkait dengan bahaya keamanan pangan -Bermanfaat menunjukkan kualitas - Masa simpan, kontaminasi, dan status higienis pada saat proses produksi Makanan dalam kaleng : ALT anaerob dan ALT aerob dimaksudkan untuk menunjukkan kontaminasi pasca proses pengalengan
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Nilai Angka Lempeng Total yang tinggi

Menunjukkan bahan baku yang terkontaminasi Sanitasi yang tidak memadai Proses pengolahan (produksi) yang tidak sempurna serta kondisi penyimpanan yang tidak baik

Nilai ALT 106 -108

mikroorganisme per gram sampel

Menunjukkan kemungkinan telah terjadi kerusakan atau produk mengalami dekomposisi.

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PERHITUNGAN KOLONI BAKTERI (1)

Dipilih cawan petri dari satu pengenceran yang menunjukkan

jumlah koloni antara 25-250 koloni Pengenceran 10-2 Cawan I 150 Cawan II 300 Keterangan Dipilih koloni cawan I

10-3

20

25

Dipilih koloni cawan II

Jumlah koloni rata-rata Jumlah kedua cawan dikalikan

dengan faktor pengencerannya Maka Angka Lempeng Total adalah : 150 + 250 x 102 = 200 x 102 2
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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
Pengenceran 10-2 Cawan I 200 Cawan II 300 Jumlah Koloni Rata-rata 250 x 102

10-3

15

25

20 x 103

Maka Angka Lempeng Total adalah :

250 + 200 x 102 = 225 x 102 2

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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
Pengenceran Cawan I Cawan II

Jumlah Koloni Rata-rata

10-2 10-3

215 26

225 30

220 x 102 28 x 103

Maka Angka Lempeng Total adalah :

220 + 280 x 102 = 250 x 102 2

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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
Pengenceran 10-2 Jumlah Koloni Rata-rata 140

10-3

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Maka Angka Lempeng Total adalah 140x102

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PERHITUNGAN KOLONI BAKTERI (5)

Bila hasil perhitungan pada tingkat pengenceran lebih tinggi diperoleh jumlah koloni rata-rata 2 kali jumlah rata-rata pada pengenceran di bawahnya, maka dihitung dari rata-rata jumlah koloni kedua tingkat pengenceran tersebut.
Pengenceran
10-2 10-3

Jumlah Koloni Rata-rata

240 41

Maka Angka Lempeng Total adalah : 240 + 410 x 102 = 325 x 102 2

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PERHITUNGAN KOLONI BAKTERI (6)

Bila tidak satupun koloni tumbuh dalam cawan, maka Angka

Lempeng Total dinyatakan sebagai <1 dikalikan faktor pengenceran terendah.

Pengenceran 10-2 Jumlah Koloni 0

10-3

Maka Angka Lempeng Total adalah <1x 102

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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

Pengenceran

Cawan I (1 Sektor)

Cawan II (1 Sektor)

10-2
10-3

100
175

150
200

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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 Pengenceran 10-2 Cawan I Jumlah koloni 100 x 4 = 400 Jumlah koloni 175 x 4 = 700 Cawan II Jumlah koloni 150 x 4 = 600 Jumlah koloni 200 x 4 = 800 Jumlah Koloni Rata-rata 500 x 102

10-3

750 x 103

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PERHITUNGAN KOLONI BAKTERI (8)

Jika jumlah koloni rata-rata dari 1/8 bagian cawan 200, maka

Angka Lempeng Total dinyatakan 200x8 dikalikan faktor pengenceran

Pengenceran

Cawan I (1 Sektor)

Cawan II (1 Sektor)

10-2
10-3

253
373

278
423

Maka Angka Lempeng Total adalah 200x8 102

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PENCATATAN HASIL ALT Penghitungan dan pencatatan hasil ditulis dalam dua angka Angka berikutnya dibulatkan ke bawah bila < 5 dan dibulatkan ke atas apabila > 5
Sebagai contoh : 523 x 103 dibulatkan menjadi 52 x 104 83,6 x 103 dibulatkan menjadi 84 x 103

Hasil dinyatakan dalam tiap gram atau tiap mL sampel

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