Presentation 1 - Ex 4

EXERCISE 4 • To determine the bacterial population using different direct and indirect methods of quantification • To interpret a growth curve • To understand the limitations of the different methods used in estimating microbial populations

DETERMINING MICROBIAL GROWTH

MICROBIAL GROWTH • GROWTH = ORDERLY INCREASE IN THE QUANTITY OF ALL THE COMPONENTS OF AN ORGANISM • BINARY FISSION (2 CELLS ARISE FROM 1 CELL) • TIME REQUIRED FOR A COMPLETE GROWTH CYCLE : VARIABLE – NUTRITIONAL – GENETIC – e.g. E. COLI (20 MINUTES – 1 CYCLE)

CELLULAR GROWTH VS POPULATION GROWTH • CELLULAR GROWTH = SYNTHESIS OF VITAL COMPONENTS (RIBOSOSMES, MEMBRANES, GENETIC MATERIAL, ETC) • POPULATION GROWTH = INCREASE OF THE NUMBER OF CELLS IN THE POPULATION (RATE AFFECTED BY ENVIRONMENT)

POPULATION GROWTH •

GROWTH RATE – CHANGE IN CELL NUMBER OR CELL MASS PER UNIT TIME

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GENERATION TIME – INTERVAL FOR THE FORMATION OF 2 CELLS FROM ONE – DOUBLING TIME

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EXPONENTIAL GROWTH – PATTERN OF POPULATION INCREASE, WHERE THE NUMBER OF CELLS DOUBLES DURING EACH UNIT TIME PERIOD

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GROWTH CYCLE – – – –

LAG LOG STATIONARY DEATH

CALCULATING THE GENERATION TIME • •

N = NO2n WHERE: – N = THE CELL NUMBER AFTER A PERIOD OF GROWTH – NO = INITIAL NUMBER OF CELLS – n= NUMBER OF GENERATIONS

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CAN BE RE-EXPRESSED IN TERMS OF n – n = LOG (N) – LOG (NO) 0.301

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GENERATION TIME = t/n

EXAMPLE • • • •

Given an initial density of 4 x 104 After 2 hours the cell density became 1 x 106 Compute for the generation time Solution: – t=2 – n = log (1 x 106) – log (4 x 104) 0.301 – n = 4.65 – Generation time = 2/4.65 or 0.43 hours (t/n)

METHODS OF QUANTIFICATION

IN OUR EXPERIMENT… • DIRECT MICROSCOPIC COUNT • TURBIDIMETRIC MEASUREMENTS • VIABLE CELL COUNT • BACTERIAL BIOMASS

DIRECT MICROSCOPIC COUNT • SPECIAL SLIDE + OVERNIGHT CULTURE • FAST AND EASY • TOTAL CELL COUNT (DEAD + LIVE) • CELLS/ML = AVERAGE NUMBER OF CELLS AREA OF MICROSCOPIC FIELD

X 100mm2 0.01ml

LIMITATIONS •

DEAD CELLS NOT DISTINGUISED FROM LIVING CELLS

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SMALL CELLS DIFFICULT TO SEE, THUS SOME CELLS MISSED

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PRECISION DIFFICULT TO ACHIEVE

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PHASE CONTRAST MICROSCOPE SOMETIMES REQUIRED (IF SAMPLE UNSTAINED)

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NOT SUITABLE FOR CELLS OF LOW DENSITY (CELLS SHOULD BE GREATER THAN 106 CELLS/ML

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DILUTE SUSPENSIONS: RECONSTITUTE FIRST BEFORE COUNTING

TURBIDIMETRIC MEASUREMENTS • ESTIMATES OF CELL MASS RATHER THAN ACTUAL NUMBER • CELL MASS PROPORTIONAL TO CELL NUMBER • THE MORE CELLS PRESENT, THE MORE LIGHT SCATTERED, MORE TURBID THE SUSPENSION • SPECTROPHOTOMETER

LIMITATIONS • NOT FOR FUNGAL SAMPLES OR SAMPLES WITH FILAMENTOUS GROWTH • AT HIGH CONCENTRATIONS OF CELLS, LIGHT SCATTERED AWAY FROM DETECTING UNIT (MAY LOOSE LINEARITY) • CAN YOU NAME OTHERS?

VIABLE CELL COUNT • ONLY LIVE CELLS COUNTED (DIVIDES AND FORMS OFFSPRINGS) • THROUGH COLONY FORMATION • “EACH VIABLE CELL CAN YIELD ONE COLONY” • THE IMPORTANCE OF DILUTION • WAYS – SPREAD PLATING – POUR PLATING – MILES AND MISRA

SOURCES OF ERRORS IN PLATING • INOCULUM SIZE • SUITABILITY OF CULTURE MEDIUM • INCUBATION CONDITION USED • LENGTH OF INCUBATION

WHY CHOOSE THE VIABLE CELL COUNT OVER THE OTHERS? • GIVES BEST INFORMATION ON THE NUMBER OF VIABLE CELLS • APPLICATIONS – FOOD AND INDUSTRY – MEDICAL – AQUATIC

• HIGH SENSITIVITY – FEW CELLS MAY BE COUNTED – DETECTION OF CONTAMINANTS – USE OF HIGHLY SELECTIVE MEDIA (MIXED POPULATIONS)

APPLICATIONS cfu/ml = AVERAGE NUMBER OF COLONIES COUNTED VOLUME PLATED

X

DF

WHAT IF THE COLONY COUNTS FALLS OUTSIDE THE ACCEPTABLE RANGE? • ACCEPTABLE RANGE – 3O-300 (SPREAD/POUR) – 10-20 (MILES AND MISRA)

• EAPC OR EHPC – ESTIMATED AEROBIC/HETEROTROPHIC PLATE COUNT

• TNTC – TOO NUMEROUS TO COUNT

• ZERO COLONIES – NO COLONIES GREW

CASE 1:1 DILUTION VALID • Given: (spread plating) – 100 = 585/612 – 10-1 = 64/79 – 10-2 = 8/5

• Solution cfu/ml = (64 + 79)/2 x 0.1 ml = 8, 540 cfu/ml

101

CASE 2:

2 DILUTIONS VALID

• Given: (spread plating) – 100 = 982/1025 – 10-1 = 158/190 – 10-2 = 38/45

• Solution • cfu/ml = (158 + 190 + 39 + 45)/4 x 0.1 ml = 10, 800 cfu/ml

101

CASE 3: NO DILUTIONS VALID (TNTC) • Given: (spread plating) – 100 = TNTC/TNTC – 10-1 = TNTC/TNTC – 10-2 = TNTC/TNTC

• EST > 1.0 X 103

CASE 4: NO DILUTIONS VALID (NO COLONIES OBSERVED) • Given: (spread plating) – 100 = 0/0 – 10-1 = 0/0 – 10-2 = 0/0

• EST < 10

BACTERIAL BIOMASS • INDIRECT • BIOMASS α CELL MASS • CELL MASS α CELL NUMBER • CELL NUMBER = GROWTH • LIMITATIONS: – NOT ACCURATE DUE TO OVERESTIMATION

INTERPRETING A GROWTH CURVE

IMPORTANCE OF GROWTH CURVE • MICROBIAL CONTROL • INFECTION • FOOD MICROBIOLOGY • CULTURAL TECHNOLOGY

FACTORS THAT LIMITS OR FAVORS THE GROWTH TO PROCEED • NUTRIENT CONCENTRATION • OTHER PARAMETERS (TO BE DISCUSSED DURING THE NEXT EXERCISE) – TEMPERATURE – ACIDITY/ALKALINITY – WATER AVAILABILITY – OXYGEN

MOST PROBABLE NUMBER (INDIRECT) • PARTICULARLY APPLICABLE FOR FOOD AND WATER TESTING • MULTIPLE TUBE FERMENTATION METHOD • BASED ON GAS PRODUCTION • ASSUMPTION : CELL WHEN PRESENT GAS WILL BE PRODUCED • ESTIMATED VIA AN MPN TABLE

METHYLENE BLUE REDUCTION TEST (INDIRECT)

• Based on the fact that the color imparted to milk by the addition of a dye such as methylene blue will disappear more or less quickly • Removal of the oxygen from milk and the formation of reducing substances during bacterial metabolism causes the color to disappear • Responsible for the oxygen consumption are the bacteria

METHYLENE BLUE REDUCTION TEST • The greater the number of bacteria in milk, the quicker will the oxygen be consumed, and in turn the sooner will the color disappear • The time of reduction is taken as a measure of the number of organisms in milk although actually it is likely that it is more truly a measure of the total metabolic reactions proceeding at the cell surface of the bacteria

MATERIALS • Methylene blue thiocyanate • For example, if the sample were still blue after 5 hours but was decolorized (white) at the 2.5 hour reading, the methylene blue reduction time would be recorded as 2 hours. • Decolorization is considered complete when four-fifths of the color has disappeared.

CLASSIFICATION • Class 1. Excellent, not decolorized in hours.

8

• Class 2. Good, decolorized in less than hours but not less than 6 hours.

8

• Class 3. Fair, decolorized in less than but not less than 2 hours.

6 hours

• Class 4. Poor, decolorized in less than hours.

2

LIMITATIONS • Low correlation with other bacterial procedures – true particularly in those samples which show extensive multiplication of the psychrotropic species

• Any manipulation that increases the oxygen affects the test – Cold milk holds more oxygen than warm milk; pouring milk back and forth from one container to another increases the amount, and at milking time much oxygen may be absorbed

• The kind of organisms affect the rate of reduction – The coliforms appear to be the most rapidly reducing organisms, closely followed by Streptococcus lactis, some of the faecal Streptococci, and certain micrococci

• Light hastens reduction and therefore the tests should be kept covered

LIMITATIONS •

The concentration of the dye should be uniform as an increased concentration lengthens the time of reduction

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Increasing the incubation temperature augments the activity of the bacteria and therefore shortens the reduction time.

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The creaming of the test samples causes a number of organisms to be removed from the body of the milk and brought to the surface with the rising fat. – This factor causes variations in the reduction time, since the bacteria are not evenly distributed.

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The accuracy of the test is increased, reduction time shortened and decolorization more uniform if the samples are periodically inverted during incubation