MENGHITUNG JUMLAH SEL

• Hitunglah jumlah mikroorganisme dalam susu

• Diketahui : 0,1ml susu diratakan dalam luasan 1 x 2 cm .
  
• Data yang diperoleh sbb.:
• Diameter bidang pandang = 0, 18 mm
• Pengamatan jumlah sel dilakukan sebanyak 10 x dengan data sbb.:

• Hitung jml. sel /ml susu. à 2.9 10⁶ sel / ml

• Rata2 jumlah sel / bir pandang = 36,8 sel
• Luas bid pandang = 3,14 x 0 09 x 0,09 mm2 = 0,025 mm2 à 36,8 sel
• 1 x 2 cm = 2 cm2 = 200 mm2 à = 200/0.025 x 36,8 sel = 294.400 sel
• Jadi 0,1 ml = 294.400 sel
• 1 ml = 1 / 0,1 x 294.400 sel à 2,9 . 10⁶ sel / ml

• Perhitungan jml mikroorganisme secara tidak langsung dengan pour plate :

• Hitung jumlah sel / ml

• Kesimpulan

• Perbandingan :

136. 10⁶ à >2

24,2 . 10⁶

• Jadi jml sel /ml = 2,42 . 10⁷

50. 10⁵ à <2

36 . 10⁶

• Jadi jml sel /ml = ( 50 . 10⁵ + 360. 10⁵ )/2 = 2,05. 10⁷

RELATED PROTOCOLS:

• Pour Plate Technique for Bacterial Enumeration
• Dilutions

• Fresh food will typically have very low bacterial content, but as it is handled and stored, the bacterial concentration may increase dramatically.
  
• Pasteurized Grade A milk is required to have less than 20,000 bacteria/mL by standard plate count.
  
• Ground beef may contain up to 50 million bacteria/gm.
  
• Since the number of bacteria may vary by several orders of magnitude, samples of these foodstuffs must be diluted and several dilutions plated out in order to achieve the desired range of colonies per plate (50-500).
  
• Typically, for pour plate technique, 1.0 mL of dilutions ranging from undiluted to 10³ (or higher) should be plated.
  
• (Greater dilution is necessary for highly contaminated samples.) The following procedure is for that purpose:

SUPPLIES:

1. Milk to be tested. Have date of origin, if possible. Calculate age of material.
2. Standard Plate count agar^*
3. Sterile dH₂O in 4 repipets
4. Clean sterile petri dishes

EQUIPMENT:

15 mL melted Plate Count Agar in:

• sterile capped 16x150mm test tubes
• 45^o C bath (deep enough to = agar depth. Hot Block, or water bath)
• vortex
• paper towel
• balance
• stainless steel spatula in test tube with 95% EtOH
• sterile 16x150mm test tubes
• 0.1, 1.0, 2.0 & 10 mL pipets, sterile
• flame
• colony counter with magnifying glass

DILUTIONS, AND ALIQUOT ADDITION TO PLATES:


Label two empty plates with your initials, the date, specimen (milk), aliquot volume (0.1 or 1.0 mL) and dilution factor (10²). Prepare dilution blank: Add 9.9 mL sterile dH₂O to a sterile 16x150mm test tube.

Dilute the milk: pipet 0.1 mL milk into above dilution tube (10² dilution), vortex to mix.

Add the aliquots: Using a 2.0 mL pipet, pipet 0.1 mL into first plate, 1.0 into the second.

Pick up a tube of 15 mL melted plate count agar, cooled to 45^oC, (here in a hot block, a 45^oC water bath can be used.)

ADD MELTED AGAR TO MAKE POUR PLATE:

Add melted agar (dry off if maintained in hot water) to each plate in turn, swirl to mix completely. Plunge the emptied tube immediately into warm water before agar solidifies to ease cleansing.

When the agar is solid, invert the plate and incubate 35^o C for 48 hr.

Count colonies on the plates and calculate CFU per mL:

CFU on plate x dil'n factor (10²) x aliquot factor (either 1/1.0 ml or 1/0.1 mL) = CFU/ mL milk

Example plate at left: 40 colonies on plate x 10² x 1/1.0 mL = 4000 CFU/mL of milk

• Enter your results into the class table ( your initials, the milk manufacturer, its expiration date, CFU/mL)
• Standard Plate Count Agar: 5 g tryptone, 2.5 g yeast ext..1 g dextrose, 15 g agar, 1 L water
• Suggested stations (at least two each? If enough repipets):
  

Empty plates station, wax pencil

• sterile bags of empty plates
• wax pencils
• 16x150 mm sterile capped test tubes
  
• test tube racks to hold 16x150 tubes (one per student)

Milk Dilution Station:

• milk
• sterile capped 16x150 mm test tubes
• repipet with sterile dH₂O, set for 9.9 mL
• vortex
• flame
• sterile pipets:
• 0.1, one per student
• 2.0, one per student

Pour Plate Station:

• hot block with styrofoam cage, 45 C
• flame
• wash tub with warm water in it.
• 16x150mm test tubes with 15 mL 45 C melted Standard Plate Count Agar (5 per student)
• paper towel
• Be sure to have enough tubes of SPCA (at least 3 tubes/person if they work in pairs on both experiments).
  

Plate count agar:

• 5 g tryptone
• 2.5 g Yeast est
• 1 g dextrose
• 15 g agar
• 1 L water (pH 7