CHAPTER II

THE EXAMINATION OF THE TOTAL BACTERIA WITH DOUBLE TUBE PROCEDURES

I.

Purpose
a. Students able to know the procedures or way identification bacteria in water
b. Students capable of understand the principle calculation the amount of bacteria in
water

II.

Introduction
Microorganisms is very small creature and can only be seen under a microscope.
One kind of microorganism is bacteria .Bacteria are unicellular organisms who
growing in the manner of binary fission they are one cells divide symmetrically. To
ease the calculation of colonies required knowledge of morphology bacteria so the
media growth to be used in conformity with nature the bacteria is. The presence of
microbes on food can be is profit or hurt. Is the results of metabolism species
microbes certain at food are needed and popular by human. The quantitative analysis
microbiology on the material food important to know the quality of foodstuffs, and
the process of to be applied on the material the food. There are several ways to
measure or counting microbes namely by calculation the number of cells, calculation
mass of cells directly, and estimation mass of cells indirectly. Calculation the number
of cells can be done by 3 methods namely by the ordinal microscopic, mpn (most
probable number), and the ordinal the dish (brady, 1999).
The growth of microorganisms that form colonies can be considered that every
colony that grows derived from a single cell, then by counting the number of a colony
it can be seen the spread of bacteria that there are on the material. The number of
microbes on an ingredient can be calculated in a great variety of ways, depends on the
material and the type of the microbes. There are two kinds of ways calculation or the
number of microbes bacteria, of the calculation of directly and indirectly. The number
of microbes calculation directly which is the amount of microbes calculated as a
whole, either dead or living, while the number of miroba calculation indirectly which
is the amount of microbes counted in the overall good of the dead or living or just for
determining the amount of microbes that are only living, this hanging in ways which
used (Brady, 1999)

III.

Fundamental Principle of the Theory

III.1
Bacteria Escherichia coli
Escherichia coli is gram-negative bacteria rod-shaped short having length
of about 2 m, the diameter of 0.7 m, wide 0,4-0,7 m and is anaerobic
facultative. E. coli form colonies round, convex, and to smooth with the edge of a
clear smith-keary, 1988; jawetz et al., 1995).
E. coli is a member of flora normal the intestines. E. Coli play an
important role in the synthesis vitamin k, conversion pigmen-pigmen bile, his

stomach acids bile and absorption of nutrients. E. Coli included in bacteria

heterotrophic who food for of substance oganik from the environment because
they cannot arrange their own of organic substances he wanted here. An organic
substance obtained from the rest of other organisms. This bacteria outlines
substance organic matter in food into inorganic substances, namely co2, h2o,
energy, and minerals. In the, these decay bacteria serve as decomposers and
providers nutrients to of plants (Ganiswarna, 1995).
E.coli be pathogenic if the number of this bacteria in the digestive tract
increasing or is outside the intestines. E. coli produce enterotoxin leading some
diarrhea cases. E. coli associated with enteropatogenic produce enterotoxin on
cells epithelial (Jawetz et al., 1995).
Characteristic of bacteria E. coli among others is aerobic or anaerobic
facultative, including into gram-negative bacteria, do not form spores, and can
ferment lactose to produce acid and gas at a temperature 35 oC - 37oC (Astuti,
2014).
III.2
A Method of Calculation Bacteria
1. Directly Calculation Bacteria
Directly Calculation Bacteria is calculation that counts the number of total
cells (a cell death and life which is with sample). Profit this method is its
implementation rapid and not need a lot of equipment. But weaknesses as the
cells that dead indistinguishable from living cell. Therefore them as. In other
words the results is the total number of cells that is in in the population.
A method of directly calculation bacteria of them that is:
a. Total Plate Count
A method of the ordinal the dish use the assumption that each cell
will live develop into a colony .The number of colony appear to be
index for the number of organisms contained in the. Technique that
needs the ability do dilution. Dishes will be incubated and then
calculated the number of colony formed.

b. Petroff-Hauser Method
Directly calculation can be done in a microscopic manner that is by
counting the number of bacteria in a unit of the contents of which are
very small. An instrument used is petroff-hauser chamber or
haemocytometer.
Haemocytometer is a calculation method in a microscopic manner.
Space count consisting of 9 large boxes with broad 1 mm 2 . One large
box in the middle, divided into 25 boxes being with long 0.05 mm. A
box being divided again to 16 small box. Thus a box of these great
contains 400 small boxes. Thick of space count this is 0.1 mm.
Bacterial cells are suspended will fill volume space count so the the
amount of bacteria per unit volume can be known.

2. Indirectly Calculation Bacteria

Indirectly calculation used to determine the number of microbes the
overall good of living and or dead only for determining the amount of that
only living depends on how to use.
A method of indirectly calculation bacteria of them that is:
a. Plate Count Method
Plate count method done by means of dissolving sample original in
various serial dissolving. Then every the results of dissolving in scattered
in jelly, with technique pour plate or streak plate. After that, incubation
done and colonies in observed in the jelly dish and counted as the total
number of colony form a unit (cfu = coloni forming units) (Goldman and
Green, 2009:18).
There are two methods used in plate count, namely:
1. Spread-plate method , the volume has usually 0.1 ml or less than a
whole the culture that in encerkan excess of spread-plate method s a
calculation easy to do because colony calculated must be growing in a
surface ( aerobic ) .Deprived of spread-plate method is the volume
used must not over 0.1 ml because can cause spreader , so that the
calculation will difficult.
2. Pour-plate method , the volume of commonly used is 0.1 - 1 ml from
culture to put on in a petri dish. Then, the dish in incubation until the
colony appears. Excess of pour-plate method is volume sample can
reach 1 ml. A deficiency of pour-plate method is an organism will
calculated their number must be strong faced temperatures of jelly.
Besides, observation calculation must also be observed carefully, for
colony can grow inside medium also (aerobic and anaerobic)
(Madigan, dkk., 2012: 129-130).
Overall, excess of the plate count is to produce estimation total
bacterial cells alive.In addition, this technique also has a sensitivity on
contamination by microbiology in food and other materials.The rest is can
occur many mistakes caused by inconsistency for plating, pipetting
inaccurate, and many other factors.
b. Most Probable Number (MPN)
Most probable number (MPN) in health people from microbiology
food, widely used to calculate the amount of bacteria is in foodstuffs. The
media is widely used to calculate patogenic bacteria a small amount of
contained in foodstuffs. This method based on dilution. When a solution
containing sel-sel microorganisms diluted continuous, will eventually
obtained a solution where there were no cells which is said to be sterile
(Buckle dkk, 1985).

MPN method used medium liquid in in a reaction tube, where

calculation were based on on the number of tube positive that is being
overgrown by microorganisms after incubation on temperature and a given
time. Observation tube positive can be seen by observing the cloudiness,
or the in gas a small tube (the durham tubes) that is put on an upside down
position , which is to microorganisms the formation of gas. For each
dilution in general is three know five series tube. More many tubes used
show precision higher, but instrument glass used also more (Fardiaz,
1992).
Assumptions that are applied in the methods of MPN is:
1. Bacteria distributed perfect in sample
2. A bacterial cell fragmentary individually, not in the form of a chain or
assemblage (coliform bacteria including E. coli separate perfect each
of his cell and does not form a chain)
3. Selected media have been appropriate for the growth of bacteria in
temperature the target and time incubation certain so that at least one
living cells capable of producing a tube positive during the period of
incubation
4. The number obtained describe the bacteria that live (viable) course. A
cell that wounded and not able to produce positive tube will not be
detected.

c. Turbidimetri

Turbidimetri is the method quick to count the amount of bacteria in

a solution use of the spectrophotometer. Bacteria absorb light comparable
to the volume of total cells (determined by size and the quantity of). When
microbes increasing or the bigger size in scattered liquid, increase in
scattered cloudiness. Cloudiness can be called optical density (absorption
light, usually measured at wavelengths 520 nm - 700 nm). To microbes
certain, a curve the standard could shows the number of organisms/ml
(determined with the methods the ordinal the cup to the measurement of
optical density (determined with of the spectrophotometer).
IV.

V.

Tools and Materials

1. Medium Lactose (milk)
2. Sample
3. Aquades
4. Reaction tube
5. Matches

6. Measuring pipette
7. Incubator
8. Bunsen burner
9. Alcohol
10. Label

Way of Working
1. Prepare tools and material
2. Prepared 100 ml water sample to be tested. Prepare also 3 reaction tubes are filled
9 ml aquades and 9 reaction tube each already contains 3 ml medium lactose
(milk)
3. Added water sample by one ml by using pipet into a tube who already contains
aquades in a tube dilution 1: 10, then whisk that mixed in homogeneous. Give
label in a tube reaction.
4. Adding 1 ml water sample from dilution 1: 10 into a dilution tube 1:100, then
whisk so that all mixed in homogeneous. Give a label on reaction tube.
5. Adding 1 ml samples from dilution 1:100 into a dilution tube 1:1000, then whisk
so that all mixed in homogeneous. Give a label on reaction tube.
6. 9 tube preparing the examination of a reaction that has been contains 3 departures
ml medium lactose ( milk). Adding each 1 ml water sample of a dilution tube 1:
10 into three tube medium lactose and each put a durham tube then closed. Give
a label on reaction tube.
7. Adding each 1 ml water sample of a dilution tube 1:100 into three tube medium
lactose and each put a durham tube then closed. Give a label on reaction tube.
8. Adding each 1 ml water sample of a dilution tube 1:1000 into three tube medium
lactose and each put a durham tube then closed. Give a label on reaction tube.

9. Incubation all reaction tubes into an incubator with the temperature 35,5oC during
1x24 hours. Should there be gas in a durham tube at the bottom, it can be
identified bacteria E . coli existing in samples of the water by using MPN table.
10. Observe and note the results

CHAPTER III
THE INFLUENCE OF TEMPERATURE ON THE GROWTH OF BACTERIA
I.
Purpose
a. Students capable of know the types of bacteria in accordance with characteristic
temperature environment.
b. Students capable of understand the principle identification bacteria in lab work this.
II.

Introduction
Life all living things independent of the environmental about, good environment
and abiotic biotic. Similarly life microorganisms, depending upon them on the
environment.Microorganisms this is not able to control factors outside fully, so her
life at all depends on the circumstances of around him. The only way to maintain his
life is conform or adapt to the environment. Adjustment self can occur quickly and
temporary time will be but can also evidence for changes that permanent so as to
affect the form of and morphology and the properties of physiology that hereditary.
Bacteria can also affect ph medium where he life. As for factors in the environment
can be divided over factors biotic and abiotic.
These factors of in being temperature play an important role in growth and
development a microorganisms, and so we need to know so done this experiment.

III.

Fundamental Principle of the Theory

Growth defined as increasing all basic elements chemical cells. This is a process
that need replication the whole of a structure, organel, and components of protoplasm
cellular with the nutrients in environment. In the growth of bacteria, all substance
essential shall be available to synthesis and maintenance of protoplasm, to the source
of energy, and environmental conditions appropriate.
Bacterial growth is increasing the number of or total mass of cells that exceeds
inoculum. Growth microbes was a volume and cell size and as increase the number of
cells. In bacteria or one-celled organism growth in more interpreted as growth
colonies, namely increase the number of colonies, size colonies an increasingly large
or subtansi or mass of bacteria in the colony the more. Growth in bacteria are defined
as increase the number of cells microbes or bacteria itself. Growth in general depends
on the condition of food and also environment. If the condition food and environment
suitable for these microorganisms so microorganisms going to grow with a relatively
short time and perfect.
Cell growth bacteria usually following a pattern certain growth in the form of a
curve sigmoidal growth. Change slope on a curve indicates transition from one phase
the development of to phase other. A curve the growth of bacteria be separated into
four phase major: phase lag (phase sluggish or lag phase), phase an exponential
growth (phase rapid growth or logs phase), phase stationer (phase static or stationary
phase) and phase the decline in of the population (decline). Phases these reflect the

state of bacteria in culture at a particular time. In between any phase there are a
period of transition the time can be passed before all cells enter its phase new.
a. Phase lag
Is phase bacteria adapt to its environment new .In this phase it bacteria have
not reached growth maximum.
b. phase logs / growth an exponential
Is phase growth reached maximum .In this phase it an increasing number of.
c. phase stationary
Is phase growth reached a point is zero .In this phase it does not occur increase
in the number of bacterial cells.
d. phase the population or phase death
In this phase it cells stop increase itself and the average death increased.
The bacteria in their activities influenced by factors environment divided into two
parts, namely factors abiotic covering chemistry and physics as well as the biotic that
deals with other living creatures. Factors chemical includes pH, DO, ammonia, and
others. While factors physics includes temperature, salinitas, osmotic pressure, drying,
and others. Any bacterium has endurance sensitivity to temperature different.
Temperature pertaining to the growth of bacteria being inducted into 3:
a. Temperature Minimum
That is the lowest temperature when underneath growth will be stopped.
b. Temperature Steady
Namely temperature where growth held the fastest and steady (called also temperature
incubation).
c. Temperature Maximum
Namely temperature highest when growth will be on top of it does not occur.
With respect to the temperature, so bacteria being inducted into 3:
Group
Psikrofil
Mesofil
Thermofil

Temperature
Minimum
-15C
5-10C
40C

Temperature
Steady
10C
30-37C
45-55C

Temperature
Maximum
20C
40C
60-80C

When microbes faced with high temperatures on the temperature maximum, will give
you some kinds of reaction, namely: (1) The point of death thermal, is the temperature to kill
microbial species within 10 minutes on certain conditions. (2) Time of death thermal, is time
needed to killed a microbial species on a fixed temperatures. Factors of affecting the point of
death thermal appointed time, temperatur, moisture, spore, age microbes, pH and composition
medium.
When microbes faced with low temperatures can cause of impaired metabolism. Read are
(1) Cold shock, is a reduction temperatures suddenly causing death bacteria, especially in

bacteria young or in phase logarithmic, (2) Freezing, include breakdowns cells with the ice
crystals in the water intracellula, (3) Lyofilisasi, is the process of cooling under the freezing
point in a state of a vacuum in terraced. This process can be used to preserve microbes
because the water of protoplasm directly volatilized without going through liquid phase in
(sublimation).
IV.

Tools and Materials

Tools:
1. Incubator

3. Reaction tube

5. Bunsen burner

2. Autoclave

4. Petri dish

6. Pipette

Materials:
1. Cotton

V.

2. Alcohol

3. Jelly nutrient

4. Sample

Way of Working
1.
Prepare tools and material.
2.
Take a bacteria in biofilm use pipet drops.
3.
Put on a beaker glass.
4.
Take aquades 27 ml.
5.
Enter 3 drops bacteria into tube reactions and make sure that was steril.
6.
Enter aquades nine ml in a tube reactions and make sure that was steril
7.
In the tube reaction.
8.
Do treatment above as many as three times.
9. Take water PDAM as many as 400 ml and put on a beaker glass about three a
beaker glass.
10. Place any tube reaction on each glass a beaker that contains water PDAM.
11. Do treatment:
a. In a beaker glass 1 enter ice cubes
b. In a beaker glass 2 heated
c. In a beaker glass 3 will not be treatment
Do treatment a, b,c simultaneously in the last 20 minutes, measuring temperature
which is to use thermometer for 1 minute, note the temperature.
12. Make sure that was steril tube reaction then open-close canister.
13. Open a petri dish.
14. Towards a liquid in a tube reaction to a petri dish.
15. Enter jelly nutrient that in a petri dish already beridi a liquid of a tube reaction.
16. Make sure that was steril cover a petri dish.
17. A petri dish closed and sterilized back.
18. Enter in an incubator with the temperature 37c for 24 hours.
19. Observe the results.