Microbiology Laboratories Report on Training Visitkchbi.chtf.stuba.sk/oda/doc/KHALID REPORT.pdf ·...

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Microbiology Laboratories Report on Training Visit In the framework of the project No. SAMRS 2011/01/02 “Development of human resource capacity of Kabul Polytechnic University” Funded by Bratislava 2013 Khalid “Nayab

Transcript of Microbiology Laboratories Report on Training Visitkchbi.chtf.stuba.sk/oda/doc/KHALID REPORT.pdf ·...

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Microbiology Laboratories

Report on Training Visit

In the framework of the project

No. SAMRS 2011/01/02

“Development of human resource capacity of Kabul Polytechnic

University”

Funded by

Bratislava 2013 Khalid “Nayab”

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SLOVAK UNIVERSITY OF TECHNOLOGY IN BRATISLAVA

FACULTY OF CHEMICAL AND FOOD TECHNOLOGY

DEPARTMENT OF CHEMICAL AND BIOCHEMICAL ENGINEERING

REPORT

ON MY ACADEMIC AND SCEINTIFIC ACTIVITIES IN TRAINING

COURSE AT THE SLOVAK UNIVERSITY OF TECHNOLOGY IN

BRATISLAVA

PREPARED BY: AHMAD KHALID “NAYAB”

Assistant Professor of Chemical Technology Faculty of Kabul Polytechnic University

GUIDENCE BY:

Ing. Barbora “Kaliňáková”

2013

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Acknowledgements

First of all, I would like to express my appreciations for the scientific and

educational training to Slovak University of Technology in Bratislava

(STUBA) for organizing this training and SlovakAid for their financial

sponsor.

I would like to thank Ing. Barbora Kaliňáková, Ph.D for her guidance and

cooperation during all training period. I learned lots of new things from

her which would be useful in my professional life. I would also like to

thank Doc. Ing. Juma Haydary, Ph.D from bottom of my hearth for

coordinating this program.

Best Regards

Khalid Nayab

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Table of Contents

Title Page

Introduction 5

Laboratory Exercise 1 6

Laboratory Exercise 2 8

Laboratory Exercise 3 11

Laboratory Exercise 4 14

Laboratory Exercise 5 17

Laboratory Exercise 6 21

Laboratory Exercise 7 24

Laboratory Exercise 8 28

Laboratory Exercise 9 31

Laboratory Exercise 10 34

Laboratory Exercise 11 37

Laboratory Exercise 12 39

Laboratory Exercise 13 42

Laboratory Exercise 14 45

Laboratory Exercise 15 52

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Introduction

This report contains theoretical and practical information about different

microbiology laboratory exercises.

These exercises are Preparation of culture media, Isolation of culture

from their natural sources, Isolation of pure culture, colony recognition of

bacteria; yeast; and fungi, different staining techniques; such as: wet

mount, negative, gram, and endospore staining; Influence of different

physical conditions on growth of microorganisms; such as: temperature,

pH, UV, and osmotic pressure; and quantification of cells.

Each exercise contains some brief theoretical information regarding

exercise, purpose of work, procedure, table of results (if necessary), and

questions answers about the exercise.

This was a brief information about this report, If you like to find more

about above mentioned topics, please turn page and read more what

are written in the text.

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Laboratory Exercise 1

Preparation of Culture Media

For cultivating microorganisms, we need culture media. This media can be solid or

liquid. Difference between solid and liquid media is the agar (solidifying agent) which

is added in solid media.

PURPOSE OF WORK

A. To prepare agar plates (NA, MEA)

B. To prepare slant agars (MEA)

PROCEDURE

A. The procedure of preparation of agar plates

weigh the ingredients as described

add the exact amount of distilled water by cylinder and dissolve (cultivation

flasks are filled up maximum 2/3 of the volume)

measure the pH of the nutrient medium (using pH paper) and adjust the pH to

the desired value using a solution of 3 mol / l KOH

add powdered agar (agar 2% w / vol)

close the cultivation flasks with a cotton stoppers, select the content and

cover with aluminium foil

autoclave at pressure of 120 kPa (about 121 ° C) for 20 minutes

after sterilization cool medium to 50 - 60 ° C under cold water

sterilize surface of laboratory table with disinfectant

prepare a sterile plastic Petri dishes and light up the gas burner

pour medium aseptically into Petri dishes (about 15 ml to 1 Petri dish)

allow Petri dishes to cool and solidify

store media turn upside down

B. The procedure of preparation agar slant

weigh powder nutrient medium as described

add the exact amount of distilled water by cylinder and dissolve powder

(cultivation flasks are filled up 2/3 of the volume)

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measure the pH of the nutrient medium (using pH paper) and adjust the pH to

the desired value using a solution of 3 mol / l KOH (pH 7.2 to 7.4 MPA, pH 6.4

6.6 for SLA)

add powdered agar (agar 2% w / vol)

close the cultivation flasks with a cotton stoppers

overcook medium in the microwave so that the agar in the medium is

completely dissolved

add 5 ml of overcook media to each tubes

close the tubes with a cotton stoppers, placed in a metal rack and cover with

aluminium foil

autoclave at pressure of 120 kPa (about 121 ° C) for 20 minutes

let solidify medium in tubes in an inclined position after sterilization

LABORATORY REPORT AND QUIZ

Questions:

1. What is the function of culture medium?

2. Define synthetic and natural medium

3. Why are culture media sterilized before use?

4. Why are Petri plates inverted after they cool?

5. What is sterilization and what is its significance?

6. How would you sterilize cultivation media if you do not have autoclave?

7. How to sterilize thermo labile substances?

8. Specify the conditions for sterilization of culture media prepared on laboratory

exercises.

Answers:

1- Providing nutrition and growth needy material for the microorganisms.

2- Synthetic medium is prepared from two or more compounds synthetically, but

natural medium are those natural compounds which would be suitable for the

cultivation of microorganisms.

3- To make sure it is not contaminated with any other microorganism and is

suitable for cultivation of selected microorganism.

4- To avoid destroying of colonies on the surface of the media from

accumulating of condensed water.

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5- Sterilization is the process of destroying all life forms in a certain condition

and its significance is that it ensures the media and tools sterility and

decontamination.

6- If we don’t have autoclave, we can use Tyndallization.

7- For sterilizing thermo labile materials, we can mostly use filtration.

8- In laboratory exercises, sterilization is mostly done at 121 Degrees Celsius for

20 minutes; however it can be done at different conditions related to the

degree of sterility we expect.

Laboratory Exercise 2

Isolation of Microorganisms from their natural sources and their enumeration

by standard plate count method

Microorganisms are very diverse in nature, they can be found almost in every

environment. In this laboratory exercise, we isolate microorganisms from their

natural sources and try to find their concentration in the source.

THE PRINCIPLE

The principle of the method is the isolation (extraction) of microorganisms from the

environment, the dilution of MO in appropriate medium and smears the cell

suspension on surface of Petri dishes. Microorganisms growing on agar (Petri

dishes, Petri plates) media form masses of cells called colonies. A colony is a large

number of cells on solid medium, which is visible to the naked eye as a discrete

entity.

The assumption is that each viable bacterial cell is separate from all others and will

develop into a single discrete colony. However, organism normally forms multiple cell

arrangements, such as chains, the colony-forming unit may consist of a chain of

bacteria rather than a single bacterium. Therefore, generally we determine the

number of Colony-Forming Units (CFUs) in known dilution. The number of (CFU) on

plate can be used to calculate the number of CFU per millilitre or gram of original

sample. As the concentration of microorganisms in the sample is too high, original

sample must be serial diluted with sterile saline until the bacteria are dilute enough to

count accurately. It means that the final plates should have between 30 and 300

colonies. A plate having 30-300 colonies is chosen because this range is considered

statistically significant. If there are less than 30 colonies on the plate, small errors in

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dilution technique or the presence of a few contaminants will have a drastic effect on

the final count. Likewise, if there are more than 300 colonies on the plate, there will

be poor isolation and colonies will have grown together. In Figure 3 is illustrated the

use of so-called decimal serial dilutions. Its principle is a gradual dilution of the

suspension always at 10 times lower concentration with pure saline. However, you

may use a combination of other arbitrary dilution (see laboratory report and quiz).

Figure 3- Principle of the standard plate count method.

PURPOSE OF WORK

C. To prepare dilution series of the samples : 10-1, 10-2, 10-3,10-4

D. To calculate the number of colony forming unit per ml of original sample on the

base of number of grown colonies

EQUIPMENT FOR A COUPLE OF STUDENTS

Sample of soil, sterile saline solution, sterile tubes labelled with wax pencil as 10-1

,

10-2

10-3

, 10-4

, sterile tips, micropipette, vortex, sterile spreader, agar plates

labelled with name and dilution as

10-1

, 10-2

, 10-3

, 10-4

, thermostated incubator

PROCEDURE

E. Procedure of decimal dilutions of samples

aseptically pipette 4.5 ml of sterile saline solution into 4 sterile tubes

aseptically withdraw 0.5 gr of stock solution of soil into the first tube (you

prepare dilution 10-1) and then tube homogenize with vortex mixer

Comment [M1]: L_shape stick or spreader

Comment [M2]: 0,5 g of soil

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aseptically withdraw 0.5 ml of suspension from 10-1 dilution into the second

tube (you prepare dilution 10-2) and then tube homogenize with vortex mixer

aseptically withdraw 0.5 ml of suspension from 10-2 dilution into third tube

(you prepare dilution 10-3) and then tube homogenize with vortex mixer

aseptically withdraw 0.5 ml of suspension from 10-3 dilution into the fourth

tube (you prepare dilution 10-4) and then tube homogenize with vortex mixer

aseptically dose 0.1 ml of suspension of stock solution as well as all prepared

dilutions on the surface of Petri dishes( from each dilution prepare 3 plate)

aseptically spread a suspension of microorganisms on the surface of Petri

dishes with a sterile stick and incubated in the inverted position for 48 hours at

30°C

at the end of the incubation period, record the number of CFU for each

dilution on a table in the laboratory report section

for the final calculation of CFU per ml of sample select only plate where the

number of colonies is in the range of 30-300

LABORATORY REPORT AND QUIZ

Table of results

Dilution Non diluted

10-1 10-2 10-3 10-4

Nutrition Agar

Plate 1 TMTC TMTC TMTC 11 4 Plate 2 TMTC TMTC TMTC 40 3 Average TMTC TMTC TMTC 26 4

CFU/ml TMTC TMTC TMTC

Malt Extract Agar

Plate 1 TMTC TMTC TMTC 125 2

Plate 2 TMTC TMTC TMTC 110 0 Average TMTC TMTC TMTC 118 1

CFU/ml - TMTC

B) Procedure of calculation of the number of colony forming unit per ml of

original sample

The final number of bacteria per 1 ml of drinking water or milk in the original sample

can be calculated as follows ( ml/CFU ):

D*10*pml/CFU (3)

Comment [H3]: Explain, what does “-“ mean

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ml/CFU is the total number of colony forming units per 1 ml of original sample, p

is the average number of colonies on Petri dishes in tube with a given dilution, 10 is

recalculation of the number of colony units per 1 ml, D total dilution of the sample.

Questions:

1- Calulate the density of the original culture (CFU/ml), if a culture is diluted 2 x

105-fold, and 0.2 ml is plated and gives rise to 160 colonies ?

2- Original cell suspension contain 5 * 105 cells per 1ml but only 43.2% is live.

Cells suspension is diluted according to the scheme below and plated on Petri

dishes. Calculate how many colonies growth on plate D?

Answers:

1- The density of the original culture is Colony Forming Unites per

Milliliter (CFU/ml).

2- The number of the cells which grow on plate (D) is 1500 Colonies.

Laboratory Exercise 3

Isolation of Pure Culture

Pure culture is called to that culture which is growth from a single cell. In order to

have pure culture, we must isolate them from a mixture of different microorganisms.

Here are some techniques which are suitable for isolation of pure culture.

Principle

A pure culture theoretically contains a single bacterial species. There are a number

of procedures available for the isolation of pure cultures from mixed populations. A

pure culture may be isolated by the use of special media with specific chemical or

physical agents that allow the enrichment or selection of one organism over another.

Easier methods for isolation of a pure culture include: spread appropriate diluted

culture on solid agar medium with a glass spreader or streak culture with a loop. The

purpose of spreading and streaking is to isolate individual cells (colony-forming units)

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on a nutrient medium. The streaking patterns shown in the figure below result in

continuous dilution of the mix culture to give well separated surface colonies.

Purpose of work

Isolate pure culture from mix of microorganisms.

Equipment for a couple of students

Petri plates containing nutrient agar and/or malt extract agar, loop, and mix culture

Procedure

Flame loop, cool it and take a small among from the mix culture. Spread culture on

the surface of agar plate according to figure. Striate arrows show spreading lines (or

plate sectors) and curved arrows show turning plate between individual spreading.

There is necessary to flame and cool the loop between individual spreading. End of

previous sector and start of next sector must intersect, and first and last sectors must

be unlinked.

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Laboratory report and quiz

Table of results

Group of Microorganisms

Cultivation Medium

Conditions of Cultivation

Count of Colony Types

Colony Description

Bacteria Nutrition

Agar

Temperature: 25°C

Time: 3 days Light mode:

normal

26

Diameter: 1mm Appearance:

circular Margin: entire Elevation: flat Color: white

Yeast Malt

Extract Agar

Temperature: 25°C

Time: 3 days Light mode:

normal

18

Diameter: 1.5mm Appearance:

irregular Margin: undulate Elevation: raised Color: light yellow

Questions:

1. What is pure culture?

2. What is a principle of isolation of pure culture?

3. If you did not obtain isolated colonies, what changes should you make in your

technique to ensure isolated colonies?

Answers:

1- We can call pure for a culture which has only one species of microbes in it,

and it is not contaminated with any other microbes.

2- The name describes the main principle of the “Isolation of Pure Culture”, as

we isolate pure culture of one cell from a mixture of cells with different

species. The technical principle of this method is drawing lines and continuing

drawing another one from the end of the previous after flaming the loop. In

fact by drawing each line we gradually reduce concentration of the cells on

the surface of the media, in order to isolate a cell and grow an isolate colony

from one cell.

3- There are lots of parameters which can cause the isolation process. Maybe

we didn’t do our work in aseptic condition, or maybe we haven’t drawn the line

accurately, or we have forgot loop or flamed loop…..

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Laboratory Exercise 4

Colony recognition of bacteria, yeast, and filamentous fungi

Colonies of different microorganisms have different properties. They can differ in

size, shape, arrangement, elevation, color, etc… from other, while some of them are

very similar. We can recognize some colonies macroscopically, without using any

microscope.

THE PRINCIPLE

Microbes are everywhere, they are found in the water we drink, the air we breathe,

and the earth we walk on. They live in and on our bodies. Microorganisms growing

on agar (Petri dishes, Petri plates) media form masses of cells called colonies. A

colony is a large number of cells on solid medium, which is visible to the naked eye

as a discrete entity. Colony morphology may be used as an aid to the identification of

microorganisms. Although colony morphology cannot be used as the sole identifying

criterion, it is used to recognize many common types of microorganisms. Six

parameters are normally used to describe microbial colonies. There are: overall

colony appearance, colony margin (edge), colony elevation, colony size, colony

pigmentation, and colony consistency. Most important parameters for practical

identification are:

size (useful characteristic for identification, the diameter of a representative

colony may be measured, prokaryotic cell colonies may be smaller than

eukaryotic cells colony - it is not always rule)

form (circular typical for bacteria, irregular-yeasts, filamentous-filamentous

fungai)

surface (bacterial colonies are frequently shiny and smooth, yeasts form

usually matt, rough, wrinkled or dull colonies, filamentous fungai are dry,

velvet)

consistency, texture (texture of bacterial colony is moist, mucoid, yeasts form

dry, hard, mealy colonies and fungal colonies are rigid)

color (consider the colour of the colony itself or pigment excretion to the

cultivation broth)

Bacteria and yeast colonies are usually white or cream colored. However for some

strains are characterized yellow, red or black pigments. Yellow pigments (flavin)

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create yeasts Eremothecium, Ashby and some species of the genus Candida,

Cryptococcus. Red (carotenoid) pigment form yeasts of the genus Rhodosporidium,

Rhodotorula, Sporobolomyces and some species of bacteria (Mycobacterium phlei,

some species of the genus Micrococcus, Flavobacterium, Halobacterium).

Carotenoid pigments are formed in the genus Neurospora conidia micromycetes up

under the influence of light. Black (melanin) pigments are characteristic of some

yeast genus Cryptococcus and Aureobasidium pullulans. Light brown to black,

leathery colonies form diploid yeast genus Rhodosporidium (in haploid form is red)

and brown brown to black pigments are typical for Azotobacter chroococcum and

micromycets of the family Dematiaceae. Some microorganisms secrete colored

pigments into the soil, for example Pseudomonas bacteria secrete yellow, green,

blue and fluorescent pigments (fluorescein, pyocyanin). Some species of Fusarium

produce yellow, red, green, dark blue and black pigments into the medium. After

prolonged cultivation, the mycelium color themselves. The presence of red and black

pigments in microbial cells has a protective effect against lethal effects of UV

component of sunlight. Therefore, these microbes are frequent airy contamination

(eg Micrococcus, Rhodotorula, Alternaria, Cladosporium etc.).

Morphological characteristics of colonies can be summarized as follows:

filamentous fungi colonies of are hard, dry, mealy colonies, velvet. Colonies

are very distinctive and cannot be confused with the bacteria and yeast !!!

yeasts colonies are usually larger than bacterial colonies. Yeast colonies are

dull, pasty, characteristic smell of fermentation. But yeast colonies are very

similar colonies of bacteria and can therefore be distinguished to each other

only when viewed in the microscope.

bacteria form small, shiny, soft colonies often with unpleasant odour

PURPOSE OF WORK

A) Describe colony morphology using accepted descriptive terms

B) Recognize colony of bacteria, yeasts and filamentous fungi between them

EQUIPMENT FOR A COUPLE OF STUDENTS

Petri plates containing nutrient agar and malt extract agar

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PROCEDURE

The purpose is to obtain sample of microorganism from environment on

surface of agar plate

Here are some suggestions: open agar plate to the air to 30 minutes, place a

hair on the agar or touch the plate with your fingers or cough on the plate,

wipe the surface of laboratory bench with a sterile cotton swab

Replace the lids of the Petri dishes, place the plates in an inverted position

(reducing the amount of condensation forming on the inside surface of the

Petri dish lid) and incubate the plates at laboratory temperature for a few

days.

Examine the colonies appearing on the plates. Describe at least three

different colony types using figure as a reference and fill in the following table

with description of the bacterial colonies.

LABORATORY REPORT AND QUIZ

Table of Results

Sample:

0.1ml

at: 25°C;

For: 7 days

Colony Description

Diameter Appearance Margin Elevation Color #

Colony 1 1mm Circular Entire Raised White 22

Colony 2 1.5mm Irregular Undulate Umbonate Light Yellow 6

Colony 3 5mm Circular Entire Flat Green 1

Questions:

1. How do bacterial colonies differ from fungal colonies?

2. What is a bacterial colony?

3. Are you able to distinguish bacteria from yeast only on the morphology of the

colonies?

4. Are you able to distinguish filamentous fungi from yeast only on the

morphology of the colonies?

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5. What is the function of pigments in the microbial cell?

Answers:

1- Bacterial colonies are smaller and grow just on the surface of the media, but

fungi colonies are much larger than bacterial colonies. They look like fur and

can grow above and also inside the media.

2- A group of cells which are formed from growing of one individual cell of

bacteria are called a bacterial colony.

3- In some cases, we can distinguish between bacterial and yeast colony if they

are a typical bacterial or yeast colony, but, most of the times, we cannot do

so, because they are very similar.

4- In some cases we cannot distinguish between yeast colonies and fungi

colonies, but Most of the times, we can do, because they are very different in

their morphological properties.

5- Protection of the microbial cell.

Laboratory Exercise 5

Wet Mount

“Wet Mount” is a method of observing microorganisms microscopically. This method

is used for talking about size, shape, arrangement of the cells, and movement – the

presence of flagella

THE PRINCIPLE

Wet mounts are the most common preparations used to view living microorganisms.

A small drop of an sterile water is placed on a clean slide, then suspension MO is

added aseptically and clean cover slip is placed over liquid and press down lightly.

Although wet mounts can be made rapidly, they dry up after five or ten minutes and

so are not very useful if long-term observations are required. Wet mounts is useful

for determining cellular size, shape and arrangement which is sometimes destroyed

during the staining process. Also Brownian movement or true motility with flagella

can be observed in wet mounts. Brownian movement is not true motility but rather is

movement caused by continuous vibrating motion by invisible molecules striking the

bacteria. In Brownian movement the particles and microorganisms all vibrate at

about the same rate and maintain their relative positions. Only the bacteria are truly

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motile, their movement will be over greater distances and will be multi-directional, not

just back and forth. The presence, number and arrangement of flagella are useful in

identifying bacterial species. True motile bacteria move from one position to another.

Their movement appears more directed than Brownian movement, and occasionally

the cells may roll or spin.

The dried preparation of cells is known as a smear. Smears can be prepared from

cells in a liquid culture or from agar plate or slant. When using a liquid suspension,

one loopfuls are smeared onto a glass slide and then allowed to air dry. However,

bacterial cells are small, virtually transparent and little or no detail in cell is

distinguishable when placed smear under the bright-field microscope. Hence the

smear stains in order easier viewing of cells. Stains with dyes (colouring,dyeing)

enhance the contrast and allow to view the cell more distinctly. The dye adheres to

the bacteria and morphology (shape, arrangement, structure, size) can be more

readily seen. More intense staining can be achieved by the extension of exposure

time, by increasing the temperature of dyeing or application of a mordant that

increases the interaction between the bacterial cell and the dye

In progressive staining color works as long as the preparation well stained. Then the

excess of dye is washed out with water. In regressive staining the preparation

intentionally stained intensely and then part of the dye washed out with dilute

alcohol.

Staining methods used in microbiology is divided into:

Simple stainning when a single stain or dye to create contrast between the cell and

the background is applied. Simple staining is often employed when information about

cell shape, size, and arrangement is desired.

At differential staining we apply the mixture of colors and different types of microbes

and their structures are different color. Different affinity of dyes can detect some

diagnostically significant features of microorganisms such as gram

positivity/negativity, presence of capsules and endospores, acid-fast staining

The cells in the dried smear are attached or fixed to the slide by briefly heating the

slide over a gas burner flame. This procedure is known as heat fixation. When using

colonies, a small drop of water is placed on the slide and very small amount of

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material is mixed with water to separate and suspend the cells. The suspension is

then spread out, air dried, and heat fixed. These fixed smears then can be used for

staining.

PURPOSE OF WORK

A. To prepare and observe wet mount slides.

B. To distinguish between true motility and Brownian movement.

C. To make and fix a smear.

EQUIPMENT FOR A COUPLE OF STUDENTS

Slides, cover-slides, sterile water

Culture: Bacillus subtilis, Micrococcus luteus, Saccharomyces cerevisiae…

A) PROCEDURE WET MOUNT TECHNIQUE

Place a small drop of sterile water on a clean slide using a pipette.

Suspend the small amount from the culture colony by stirring carefully.

Handle the cover slip carefully by its edge and place in on the drop.

Observe using microscope and record your observations to Table

B) PROCEDURE FIX SMEAR

place a small drop of sterile water on a clean slide

aseptically add a small amount of culture colony, stir the suspension by

inoculation loop and spread over the slide so as to create a thin film.

let air dry

heat fixes the dried smear by passing the slide through the flame three times.

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LABORATORY REPORT AND QUIZ

Table of observations

Appearance Bacillus subtilis Micrococcus

luteus Saccharomyces

cerevisiae

Draw the types of

microorganisms observed:

Shape and arrangement:

Rod shaped, convex elevation, and entire

margin

Tetrads, oval shaped, and raised

elevation Oval

Motility:

True motility, Brownian

movement, Fluid movement

Brownian movement, Fluid

movement Same like m.l.

Questions:

1. How do you distinguish true motility from Brownian movement or motion of the fluid?

2. Can you distinguish the prokaryotic organisms from the eukaryotic organisms? 3. Which of the bacteria exhibited true motility on the slides? 4. Why do you fix the smear before staining? 5. Can the light intensity of your microscope be regulated? Explain. 6. Why is oil necessary when using the 90 × to 100 × objective?

Answers:

1- Brownian movement is something like a regular shaking of the particles in the

liquid. Fluid movement is distinguished easily as it happens in a clear

direction, so the irregular movements which are not similar to Brownian

movement and fluid movement; are true motility from the microorganisms

which are movable. They have different types of motility.

2- Prokaryotic organisms have basic cell structure and much smaller cells as

eukaryotic organisms and Eukaryotic cell structures are much complicated

than prokaryotic cells.

3- Bacillus subtilis … those with flagellum.

Comment [H4]: Saccharomyces cerevisiae?

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4- For two reasons we do fix the smear. Not to lose the bacteria or yeast cells

from the surface of slide during staining and also fixing can kill the organisms

and help the microscopy process (means staining) as we can see better the

structures inside the cells.

5- Yes, intensity of the microscope light can be regulated with a related knob.

6- To make better the resolution of the view. - prevent loss of light conditions/sharp

vision through refraction of rays

Laboratory Exercise 6

Negative Staining

“Negative Staining” is another method of observing microorganisms microscopically.

Characteristic of this method is that, we do not color the microorganism, but the

background.

By coloring the background, it is much easier to observe the microorganisms,

because eyes can easily differentiate between the things which are different in

colors.

THE PRINCIPLE

The negative stain technique does not stain the bacteria but stains the background.

The bacteria will appear clear against a stained background. Since simple staining

procedures are rapid and easy to carry out, they are often used when information

about cell shape, size, and arrangement is desired.

Bacteria can generally be characterized as spheres (coccus, plural cocci), rods

(bacillus, plural bacilli), spirals (spirillum, plural spirilla), helices (spirochete, plural

spirochetes), or branched organisms. In addition, many organisms form very

distinctive arrangements that can be used to identify them. For example, bacteria

such as the streptococci form chains of cells, the staphylococci develop in grape-like

clumps, the neisseriae exist as pairs or diplococcic (diplo = pair of), and some

micrococci and sarcinae (sarcina – a package) are typically found in package of four

or eight.

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Fig. 3- Common shapes and arrangements of bacteria.

PURPOSE OF WORK:

A) To prepare a negative stain.

EQUIPMENT FOR A COUPLE OF STUDENTS

Congo red (2% solution in ethyl alcohol), slide

Culture: Micrococcus luteus and Bacillus subtilis

PROCEDURE

Slides must be clean and grease-free. Place a small drop of Congo red at the

end of the slide.

Mix a small amount of the culture on solid media in the Congo red drop-

using loop. Using the loop spread the drop out to produce a smear.

Let the smear air dry.

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Dip the completely dried smear in to the solution of chloride acid (1%) for a

few seconds (Congo red changes colour from red to blue).

Let the smear air dry again.

Examine the stained slides microscopically using the low, high-dry, and oil

immersion objectives.

LABORATORY REPORT AND QUIZ

Draw a representative field of your microscopic observation of negative staining

Appearance *Bacillus subtilis Micrococcus luteus

Magnification 40X 40X

Morphology and arrangement

Rod shaped, chains Spherical shaped, Tetrads

Questions:

1. When is negative staining used?

2. What is an advantage of negative staining?

3. Why is negative staining also called either indirect or background staining

Answers:

1- When we need information about the size, shape, and arrangement of the

cells, we can use negative staining.

2- It stains the background, not the bacteria itself and it would be easier to talk

about the colony description.

3- Because it stains the background, not the bacteria itself.

Comment [H5]: Only objective, total is 400x?

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Laboratory Exercise 7

Gram Staining

According to their cell wall composition, bacteria can be gram positive or gram

negative. Gram positive bacteria have much thicker peptidoglycan layer than gram

negative bacteria. That is why these two categories of bacteria react differently to the

gram staining which shows the gram negativity or gram positivity of them.

PRINCIPLE

The Gram stain is a very useful stain for identifying and classifying bacteria. The

Gram stain is a differential stain that allows you to classify bacteria as either gram-

positive or gram-negative. Bacteria stain differently because of chemical and

physical differences in their cell walls. The Gram-positive bacteria have a thick cell

wall that consists primarily of peptidoglycan (see figure 4). Many Gram-positive

bacteria, however, have polymers called teichoic acid in their cell walls, which may

account for as much as 50% of the wall’s weight. The peptidoglycan layer of the cell

wall consists of polysaccharides, made up of alternating N-acetylglucosamine and

N-acetylmuramic acid units, cross-linked by short peptides, while teichoic acids are

long polymers of alternating phosphates and carbohydrates (e.g., ribitol or glycerol).

The cell wall of Gram-positive bacteria is generally between 20 and 80 nanometers

thick. The walls of Gram-negative bacteria have much less peptidoglycan than those

of Gram-positive bacteria. The peptidoglycan layer, usually about 2 nanometers

thick, is surrounded by a complex lipid bilayer called the outer membrane. No

teichoic acids are associated with the cell wall of Gram-negative bacteria. The Gram

stain is most consistent when done on young culture of bacteria (less than 24 hours

old). The staining technique consists of the following steps:

Apply primary stain – crystal violet. All bacteria are stained purple by this basic dye.

Apply mordant – Gram’s iodine. The iodine combines with the crystal violet in the cell

to form a crystal violet-iodine complex.

Apply decolorizing agent – ethyl alcohol or acetone. The crystal violet is washed out

(decolorized) of some bacteria, while others are unaffected. The crystal violet –

iodine complex is larger than the crystal violet or iodine molecules that initially

entered the cell and cannot pass through thick peptidoglycan in Gram-positive

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bacteria. In Gram-negative cells, the alcohol dissolves the outer lipopolysaccharide

layer, and the complex washes out through the thin layer of peptidoglycan.

Apply secondary stain – safranin. This basic stains the decolorized bacteria red.

Fig. 4- Gram positive and Gram negative cell wall.

PURPOSE

A) Perform and interpret Gram stains.

EQUIPMENT FOR A COUPLE OF STUDENTS

Gram staining reagents: crystal violet, Gram’s iodine, ethyl alcohol, safranin, Wash

bottle of distilled water, slide, Culture: Escherichia coli and Bacillus subtilis

PROCEDURE

Prepare and fix smears.

Prepare a Gram stain. Use a clothespin or pincette to hold the slide.

Cover the smear with crystal violet and leave for 60 seconds.

Cover the smear with Gram’s iodine for 30 seconds.

Decolorize with 95% ethyl alcohol. Let the alcohol run through the smear

(usually 5 to 10 seconds). This is a critical step. Do not over decolorize.

Immediately wash gently with distilled water by tilting the slide and squirting

water above the smear so that the water runs over the smear.

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Add safranin for 60 seconds.

Wash with distilled water and let dry the slide freely in air.

Examine the staining slide microscopically using the low and oil immersion

objectives.

LABORATORY REPORT AND QUIZ

Draw a representative field of your microscopic observation of Gram staining

Appearance Bacillus subtilis Escherichia coli Micrococcus

luteus

Sketch a few bacteria.

Morphology, arrangement, relative size

Rod Shaped Rod Shaped Tetrads

Color Dark blue Red Dark blue

Gram reaction Gram Positive Gram Negative Gram Positive

Questions:

1. If you leave the alcohol on too long, what would you expect to see after Gram

staining a mixture of Escherichia coli and Bacillus subtilis?

2. When you counter stain with safranin, why do the Gram-positive bacteria not

pick up safranin and stain red?

3. Why might a physician perform a gram stain on a sample before prescribing an

antibiotic?

4. Explain the principle of Gram staining.

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Answers:

1- If we leave alcohol for too long time than usual, in our results we will have to say

that both are gram negative bacteria as both of them take red color during gram

staining.

2- Because they are already colored purple.

3- To determine if the bacteria are gram positive or negative to prescribe broad-

spectrum antibiotics.

4- Bacteria are classified according to their cell wall structure to two main groups

which are gram positive and gram negative bacteria. The peptidoglycan layers of

the gram positive bacteria are much thicker than gram negative.

After fixing the smear, first we add crystal violet for 30 to 60 seconds, then add

iodine for 30 to 60 seconds, and then throw both away and wash it wish alcohol

for 5 to 10 seconds and then cover the smear with safranin.

Crystal violet color both gram positive and negative bacteria purple, but when we

wash them with alcohol gram negative become colorless because losing crystal

violet, but gram positive bacteria have thicker layer and keep the crystal violet.

After adding safranin gram negative bacteria which are now colorless, get red

color, so this way we can determine which bacteria are gram positive and which

are gram negative.

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Laboratory Exercise 8

Endospore Staining

Endospore staining is a method of observing microorganisms microscopically. This

method is used to determine the endospores of the endospore forming bacteria.

As we know, endospores are much stronger in physical and chemical properties, as

they are used to protect bacteria in unfavorable conditions, that is why endospore

staining is much difficult process than other types of staining, and needs special

requirements and conditions.

THE PRINCIPLE

An endospore is a heat- and chemical-resistant resting form produced by member of

certain bacteria genera in response to adverse environmental condition. The only

bacteria known to produce endospores belong to the following genera: Bacillus,

Clostridium, Desulfotomaculum, Sporolactobacillus, Thermoactinomyces, and

Sporosarcina. Because of endospore’s outer coat is an effective barrier to chemicals,

endospores generally stained poorly. Endospores can be stained by using very hot

dyes. Figure represents typical life cycle of spore forming bacterium.

Fig. 5- The development cycle of Endospores.

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PURPOSE

A) Prepare and interpret endospore stain.

EQUIPMENT FOR A COUPLE OF STUDENTS:

Endospore staining reagents: malachite green, safranin, Wash bottle of distilled water,

slide, and heater

Culture: Bacillus subtilis (older than 48 hours)

PROCEDURE

Prepare and fix smears.

Prepare an endospore stain. Use a clothespin or pincette to hold the slide.

Cover the smear with malachite green.

Heat the stain. The malachite green must heat for at least 5 minutes.

Cool the slide for about 1 minute before continuing.

Wash gently with distilled water by tilting the slide and squirting water above the

smear so that the water runs over the smear.

Add safranin for 60 seconds.

Wash with distilled water and let dry the slide freely in air.

Examine the staining slide microscopically using the low and oil immersion

objectives. Label the vegetative cells and endospores.

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LABORATORY REPORT AND QUIZ

Draw a representative field of your microscopic observation of endospore staining

Appearance Bacillus subtilis

Sketch a few bacteria (1000X)

Questions:

1. How did the appearance of the 24-hour and 72-hour Bacillus subtilis culture differ?

2. Which organism (genus and species) is responsible for each of the following

diseases: anthrax, tetanus, botulism, and gas gangrene?

3. Why is heat necessary in order to stain endospores?

4. What is the function of an endospore?

5. Why are endospores so difficult to stain?

Answers:

1- 72 hours old culture has more spores than 24 hours old culture.

2- Anthrax – Bacillus anthracis

Tetanus – Clostridium tetani

Botulism – Clostridium botulinum

Gas gangrene – Clostridium perfringens

3- Because endospores are much stronger against laboratory temperatures.

4- Endospores protect bacteria from abnormal physical and chemical conditions.

5- Because of theirs strong physical structure.

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Laboratory Exercise 9

Fungi (yeast and molds)

Fungi can be yeast or molds. Yeast and mold are two different types of fungi. Both

yeast and fungi are eukaryotic organisms. Yeasts are unicellular, microscopic

microorganism, while molds are multicellular macroscopic organisms.

PURPOSE

A. Characterize and classify fungi.

B. Identify common saprophytic molds and yeasts.

C. Explain dimorphisms.

THE PRINCIPLE

Fungi possess eukaryotic cells and can exist as unicellular or multicellular organisms.

They are heterotrophic and . Fungi are aerobic, facultative anaerobic and anaerobic.

Unicellular yeasts and multicellular molds are included in the Kingdom Fungi.

Yeasts are nonfilamentous, unicellular fungi that are typically spherical or oval in shape.

They reproduce asexually by budding. In some instances, when buds fail to detach

themselves, a short chain of cells called a pseudohypha forms. Depending on the strain

and the external conditions that prevail in the culture yeast form or (pseudo)hypha form,

that is called dimorphism.

A macroscopic mold colony is called a thallus and is composed of a mass.

PURPOSE

D. Characterize and classify fungi.

E. Identify common saprophytic molds and yeasts.

F. Explain dimorphisms.

EQUIPMENT FOR A COUPLE OF STUDENTS

Petri plates containing Sabouraud agar, Methylene blue, Lactophenol Cotton Blue

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Culture: Rhodotorula glutinis, Candida utilis, Saccharomyces cerevisiae, Geotrichum

candidum

Rhizopus oryzae, Aspergillus niger, Penicillium purpurogenum

PROCEDURE

Yeasts

Make a wet mount of each culture by using a small drop methylene blue. Record your

observations.

Molds

Make tease mount wet hyphae for observing fungi and record.

LABORATORY REPORT AND QUIZ

Draw a representative field of your microscopic observation

Organism Draw a Typical Colony Wet Mount

Saccharomyces cerevisiae Color: cream

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Rhodotorula glutinis Color: red

Candida utilis Color: cream

Rhizopus oryzae Colony appearance: filamentous Macroscopic hyphae color: Light green Spore color: Light green Underside color: Light yellow

Aspergillus niger

Colony appearance: filamentous Macroscopic hyphae color: Black Spore color: Black Underside color: White

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Penicillium purpurogenom colony appearance: filamentous Macroscopic hyphae color: Light blue Spore color: green Underside color: red

Geotrichum candidum Colony appearance: f Macroscopic hyphae color: Light blue Spore color: NA Underside color: White

Laboratory Exercise 10

Rapid determination of lethal temperatures

Temperature is one of the most important physical conditions which can influence the

growth of microorganisms. There are three different groups of microorganisms

according to their reaction to the temperature, which we will discuss it later. Lethal

temperature is the lowest temperature at which microorganism cannot survive more

than 10 minutes.

THE PRINCIPLE

Cells in addition to nutrients and enough available energy must also have optimal

physical, chemical and biological conditions. Microorganisms are able to at least

partially adapt to external physical agents. Ambient temperature is the most important

factor that affects the growth of microorganisms. Temperatures that allow growth of

microorganisms are from 0 °C to 85-90 °C, although described even higher

temperatures at which certain types of bacteria grow and live. Then to evaluate the

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dependence of the growth on temperature, it is possible to determine three

temperatures: minimum is the lowest temperature at which micro-organism yet

reproduce measurable speed, optimum at which achieves the highest growth rate (μ

max) and maximum is the highest temperature at which even leads to cell division.

Psychrophiles organisms have optimum growth temperature at 15 to 20 ˚ C. No

pathogenic microorganisms are not among psychrophiles. Most microorganisms are

mesophiles and have optimum temperature 37˚C. These include, for example

Escherichia coli, Bacillus, Saccharomyces, and all pathogens. Thermophiles will grow

best at 55 to 60 ˚ C, we find here the representatives of cyanobacteria, streptococci,

lactobacilli. Usually, it is important to determine the optimum growth temperature or

lethal temperature, less frequent temperature range of growth. Lethal temperature is the

lowest temperature that kills within a certain time microorganism under precisely defined

conditions. Usually lethal temperature means the temperature at which kills the cells in

suspension for 10 minutes. Lethal temperature for different organisms varies and is

influenced by cell concentration, culture physiological state (age) composition of media

and pH. Determination of lethal time is important from a technological point of view.

Lethal time is the time required to kill the microorganisms in a given temperature and

the defined conditions. Relationship between lethal temperature and the time needed to

death organism is especially important in the canning industry.

PURPOSE OF WORK

To determine lethal temperature of various microorganisms

EQUIPMENT FOR A COUPLE OF STUDENTS

Suspension of microorganism, 8 sterile tubes with cup, agar plate, micropipette, sterile

tips, water thermostat.

PROCEDURE OF LETHAL TEMPERATURE DETERMINATION

to 7 sterile tubes aseptically add 2 ml of tested suspension

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the bottoms of the Petri dishes with agar divide into 8 pieces and marked them

from1 to 8.

set temperature of the thermostat to 40C. After temperature stabilization insert

first tube in the thermostat. After 11 minutes take tube and cools quickly for 5

minutes in an ice solution

raise the temperature of the thermostat gradually to 45, 50, 55,60,65,70,75,85C

and proceed as in the previous paragraph

inoculate 50 ml of microbial suspension from each tube to the appropriate section

of agar

for each studied microorganism determine the lowest temperature (lethal

temperature) at which no cells grow

LABORATORY REPORT AND QUIZ

1 Day old Bacillus subtilis

Temperature 20 40 50 60 70 80 90 100

Cells ++ +++ + + - - - -

14 Days old Bacillus subtilis

Temperature 20 40 50 60 70 80 90 100

Cells +++ +++ ++ ++ + + - -

Escherichia coli

Temperature 20 40 50 60 70 80 90 100

Cells ++ +++ +++ ++ ++ + + -

Questions:

For each studied microorganism determine the lowest temperature (lethal temperature)

at which no cells grow.

1. What is the minimum, optimum and maximum temperature for growth of

microorganisms?

2. Define the lethal temperature

3. How can you determine experimentally whether a bacterium is a psychrophiles

or a mesophiles?

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Answers:

Lethal temperature for one day old Bacillus subtilis is 70 degrees Celsius, for 14

days old Bacillus subtilis is 90 degrees Celsius, and for E. coli it is 100 degrees Celsius.

1- Minimum growth temperature is the lowest temperature that a microorganism can

survive.

Optimum growth temperature is the temperature in which the microorganism has

the highest growth speed.

Maximum growth temperature is the highest temperature that a microorganism

can survive.

2- Lethal temperature is the lowest temperature that a microorganism can survive

at, for more than 10 minutes and in a specific condition.

3- Psychrophiles have lower lethal and optimal temperature than mesophiles.

Laboratory Exercise 11

Influence of pH on growth

Microorganisms, like other organisms can survive and grow at a range of pH. This

range consists of minimum pH, maximum pH, and optimal pH, which is important for a

microorganism to grow.

THE PRINCIPLE

The content of hydrogen ions (pH) in the environment strongly influences the growth of

microorganisms. The concentrations of H + ions change charge of cell membrane and

thus permeability and nutrient intake. Most bacteria grow in neutral or slightly alkaline

environment. For yeast is optimal acidic environment (pH 4.8 to 5.5). The optimum pH

for most molds is near the neutral value, but generally the molds reproduce a very wide

range of p H, i.e. from 1.2 to 11.0. In many cases, the micro-organisms themselves

affect the pH of its environment using metabolism, such as the acidifying bacteria, fungi

and yeast produce organic acids and reduce the pH of the medium. Biological buffers

are used to absorb fluctuations in pH of the culture medium. In industrial bioreactors pH

was maintained by addition of concentrated solutions of alkali or acids.

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PURPOSE OF WORK

A) To determine optimum pH for the tested microorganisms

EQUIPMENT FOR A COUPLE OF STUDENTS

Suspension of microorganism (inoculum), liquid cultivation media, Na2 HPO4 *2H2O,

citric acid, pH meter, volumetric flasks 0,5 l, tubes with metal plug, spectrophotometer,

cuvettes, vortex

PROCEDURE OF OPTIMAL pH DETERMINATION

Prepare a stock solution of 0.5 l Na2 HPO4 *2H2O with s concentration of 0.2

mol/l

Prepare a stock solution of 0.5 l citric acid with concentration of 0.1 mol/l

Mix using a pH meter buffers with different pH from stock solutions of Na2 HPO4

*2H2O and citric acid

Mix the nutrient media with different pH buffers in ratio 1:1 (final concentration of

nutritives in solutions are correct) as reported in Table:

prepared culture media (different pH) sterilized in an autoclave

after sterilization, aseptically add 5 ml culture media with different pH to the

empty tubes

incubate the bacteria at 37 ° C for 24 hours and yeast at 25 ° C 48 hours

measure the absorbance of all samples against blank

LABORATORY REPORT AND QUIZ

PH MO

2 3 4 5 6 7 8 9 10

Bacillus subtilis 0.06 0.01 0.05 0.03 0.19 0.72 1.66 1.76 1.73

Esherichia coli 0.07 0.04 0.04 0.08 0.10 0.42 1.64 1.71 1.66

Saccharomyces cerevisiae

0.42 2.00 2.00 1.55 1.22 0.42 0.14 0.09 0.08

Rhudutorula glutinis

0.79 0.07 0.68 0.49 1.05 1.22 0.81 0.79 0.73

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For each studied microorganism determine optimal pH for growth

1. What is the optimal pH for growth of yeasts, bacteria and fungi?

2. Why are buffers added to culture media?

3. What is the pH tolerance of bacteria compared to yeasts?

4. How do microorganisms change the pH of their own environment?

Bacillus subtilis and E. coli can grow better in alkaline environment, but

Saccharomyces cerevisae need an acidic environment for growth, while Rodutorulla

glutinis prefer neutral pH.

1- Bacteria 6-8

Yeast 4-4.5

Fungi 4-8.5

2- To stabilize the pH and keep pH in an exact value during cultivation.

3- Yeast can tolerate more acidic environment than bacteria.

4- Microorganisms can change the pH of their environment by producing some

special compounds or interacting with the existing compounds.

Laboratory Exercise 12

Influence of osmotic pressure on cell growth

If osmotic pressures inside the cell wall and outside the cell wall are the same, the cell

is in isotonic condition and can grow normally, but these pressures can differ because of

higher or lower concentrations of the salts in two sides of the cell wall. Higher osmotic

pressures will lead to drying of cell and lower osmotic pressures will lead to the lyses of

the cell.

THE PRINCIPLE

Microorganisms grow best in an environment that has the same or only slightly

lower osmotic pressure than the cytoplasm. Osmotic pressure is the force developed

when a membrane that is permeable only to the solvent separates two solutions of

different solute concentrations. In an isotonic solution, the concentration of solutes is the

same (means equal) outside and inside the bacterium. The bacterium is in osmotic

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equilibrium with its environment and does not change volume. In hypotonic environment

(low solute, high-water content) higher osmotic pressure of the cell is to be

compensated so that the water enters through the cytoplasm membrane into the cell

and cause it to burst. On the other hand, in the hypertonic environment (high solute,

lower water content), the water leaves from cell and the drainage of cells occurs thereby

reduces the metabolic activity of cells. This effect is used for preserving food with salt or

sugar. Microorganisms that are able to grow at high salt concentration are halophiles

organisms. The so-called osmotolerant yeast are growing well in the presence of

60%sucrose, some halophiles bacteria need to his growth NaCl concentrations of 5-

20%, other so-called extreme halophiles need 30% of NaCl.

PURPOSE

Perform an experiment that relates bacterial growth to osmotic

pressure

EQUIPMENT FOR A COUPLE OF STUDENTS

24- to 48-hour broth cultures of different bacteria, wax pencil, inoculation loop, Bunsen

burner

1 Petri plate nutrient agar with 0% NaCl

1 Petri plate nutrient agar with 0.5% NaCl

1 Petri plate nutrient agar with 5% NaCl

1 Petri plate nutrient agar with 10% NaCl

1 Petri plate nutrient agar with 20% NaCl

1 Petri plate nutrient agar with 25% NaCl

PROCEDURE

With a wax pencil, divide the bottom of each of the five Petri plates into half

Place the name of the bacterium to be inoculated in each section and salt

concentration

Streak the respective bacteria onto the six different Petri plates.

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Incubate the plates, inverted, for 48 hours

Observe the relative amount of growth in each section at each salt concentration.

Record this

growth as – (none), +, ++, +++, and ++++ (the most) in the report

LABORATORY REPORT AND QUIZ

Table of observation

Media E. coli S. marcescence

B. subtilis M. luteus

0% NaCl ++++ ++++ ++++ +++

0.5% NaCl ++++ ++++ ++++ ++

5% NaCl +++ - +++ +

10% NaCl + - + -

20% NaCl - - + -

25% NaCl - - - -

Question:

1. Which of these bacteria tolerates the most salt?

2. Which of these bacteria tolerates a broad range of salt?

3. What foods can you think of that are protected from microbial destruction by

salting?

4. What is:

a. osmosis

b. osmotic pressure

c. plasmolysis

d. halophiles

Answers:

1- Bacillus subtilis

2- Bacillus subtilis

3- Foods with higher salt concentrations.

4- Osmosis – the process of transferring of small molecules through semipermeable

membrane and not transferring larger molecules.

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Osmotic Pressure – the pressure which is produced in the both sides of a

semipermeable membrane because of the difference of the concentrations

between the both sides.

Plasmolysis – Process of losing of water from inside the cell and cell drying.

Halophiles – Organisms especially microorganisms that grow in a saline

conditions.

Laboratory Exercise 13

Influence of UV

UV radiation is also one of the most important physical conditions which can influence

the growth of microorganisms. UV can stop the growth of the microorganisms or also

can kill them. In this laboratory exercise, we use different materials for protecting

microorganisms against UV radiation and compare, which ones can do protect

microorganisms from UV, and ones cannot.

Principle

Radiant energy comes to Earth from Sun and other extraterrestrial sources, and some

is generated on Earth from natural and man-made sources. Radiation differs in

wavelength and energy. The shorter wavelengths have more energy. X rays and

gamma rays are forms of ionizing radiation. Their principal effect is to ionize water into

highly reactive free radicals that can break strands of DNA. Some no ionizing

wavelengths are essential for biochemical processes, e.g. photosynthesis. No ionizing

radiation between 15 and 390 nm is called ultraviolet (UV). The most lethal

wavelengths, called germicidal, are in the range of 200 – 290 nm. These wavelengths

correspond to the optimal absorption wavelengths of DNA.

Ultraviolet light induces pyrimidine dimmers in the nucleic acid, which results in a

mutation. Mutations in critical genes result in the death of the cell unless the damage is

repaired. When pyrimidine dimmers are exposed to visible light, the enzyme pyrimidine

dimmerase is active and splits the dimmers. This is called light repair or

photoreactivation. Another repair mechanism, called dark repair, is independent of light.

Dimmers are removed by endonuclease, DNA polymerase replaces the bases, and

DNA ligase seals the sugar-phosphate backbone.

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Purpose of work

Examine the effects of ultraviolet radiation on bacteria.

Ascertain protection properties of material against ultraviolet radiation.

Equipment for a couple of students

Petri plates containing nutrient agar, sterile cotton swabs, and covers (choose three):

gauze, paper, aluminum foil, glass; ultraviolet lamp, suspension of Serratia marcescens

in saline solution

Procedure

Swab the surface of each plate with Serratia marcescens, to ensure complete covering,

swab the surface in two directions.

Remove the lid of an inoculated plate and cover one-half of the plate with one of the

covering materials.

Place each plate directly under the ultraviolet light about 30 cm from the light agar side

up.

Expose plate for 30 seconds, 60 seconds, 90 seconds, and 120 seconds and incubate

in dark at 25°C.

Laboratory report and quiz

Sketch your results, note any pigmentation.

Material Used for Covering

0 sec 30 sec 60 sec 90 sec 120 sec

Paper

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Glass

Guaze

Al foil

Questions:

1. If you use Bacillus like biological model, would it be any difference between 24 hour

and 72 hour old culture?

2. What is the practical meaning of radiation treatment?

3. Why are plates incubated in dark?

4. What was the color of Serratia colonies? Were any of them white? If yes, try to

explain it.

5. Which cover material was the best for protection before radiation?

Answers:

1- As 72 hours old Bacillus has more spores than 24 hours Bacillus and spores are

much stronger against UV than vegetative cells, then older one can survive more

in UV conditions.

2- In microbiology laboratories, the term “Radiation Treatment” refer the treatment

of the air of the environment of the laboratory with the radiations especially UV

radiation.

3- After exposing the plates (microorganisms) to the UV radiation, we incubate them

in dark, because we let the their auto-regulatory system to eliminate the

destructive influence of UV on them.

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4- Normally the color of Serratia is red, but in some cases of exposing them to the

UV; their color change to white, and it is because a mutation changes in their

structure. It doesn’t happen usually, but sometimes.

5- The materials which we used, all were good for protecting the microbes from UV

except “guaze”. Aluminum foil is the best material for protecting microbes against

UV radiation; comparing to paper, guaze, and glass.

Laboratory Exercise 14

Quantification of cell

In microbiology is often necessary to determine the number of microbial cells in a given

environment.

To quantify the number of cells is important:

in food raw materials, the finished products, the air, or water

for the control of technological processes of fermentation

in search of the optimal growing conditions for a particular type of microorganism

in monitoring the effectiveness of disinfectants, chemotherapeutic agents,

antibiotics

Direct microscopic counting in chamber is a quick and easy method, but it is impossible

to distinguish between live and dead cells. The counting chamber (Burker chamber) is a

specimen slide that is used to determine the concentration of cells in a liquid sample. It

is frequently used to determine the concentration of blood cells but also the

concentration of yeasts or bacteria in a sample. The cover glass, which is placed on the

sample, does not simply float on the liquid, but is held in place at a specified height

(usually 0.1mm). Additionally, a grid is etched into the glass of the chamber. This grid,

an arrangement of squares of different sizes, allows for an easy counting of cells.

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Fig.6- Burker chamber

This way it is possible to determine the number of cells in a specified volume. Usually

we count the number of cells on the area of 1/25 mm. Then the total number of cells per

1 ml (P/ml) of the suspension is then determined according to the following formula:

D*000250*pP (4)

where p is the average number of cells per area of 1/25 mm2, 250 000 the calculation

to 1 cm3 (i.e. 1 ml, 1/25 · 1/10 · 1/1, 000 mm3), D is overall suspense dilution.

Cultivation method is based on dilution of MO in appropriate medium and smears the

cell suspension on surface of agar. Microorganisms growing on agar form colonies and

the number of bacteria (CFU) per millilitre or gram of sample can be calculated on the

base of number of visible colony. Assuming a very small number of cells, samples were

concentrated by filtration.

Conversely, at high concentration samples are diluted 10 or 100 times. If we do not

know the approximate number of cells, we cultivate several different dilutions in order to

achieve the required number of colony i.e. 30-300. In liquid culture, the medium

appears more and cloudier as the bacteria increase in number by division. A tube of

bacteria will tend to reflect light so that less light is transmitted through the tube. A

spectrophotometer can measure the amount of light passing through the tube, or

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conversely the amount of light absorbed. These measurements of turbidity or optical

density (OD) are not direct measurements of bacterial numbers, but an indirect

measurement of cell biomass that includes both living and dead cells. As the bacterial

cell population increases, the amount of transmitted light decreases, increasing the

absorbance reading on the spectrophotometer. Based on the calibration line, which is

dependence of absorbance on cell number, the total number of cells in the unknown

sample can be calculated. Because a value of absorbed light depends on the

wavelength of the light used, cell turbidity should be measured in the range 500-600nm

when cells exhibit absorption maximum.

PURPOSE

A) To measure absorbance a bacterial culture by spectrophotometer and using a

calibration line to calculate the number of cells in suspension

B) To determine the concentration of viable cell by cultivation method on agar

plates

C) To determine the number of cells by direct microscopic counting in Burker

chamber

D) To determine the concentration of dry cell per millilitre

EQUIPMENT FOR A COUPLE OF STUDENTS

analytical balance, sterile saline solution, sterile tips, micropipette, vortex, sterile sticks,

agar plate, incubator, spectrophotometer, cuvettes, agar plate, tips, micropipette,

moisture analyser, cellulose acetate membrane 0.2 µm

A) PROCEDURE OF SPECTROPHOTOMETRIC DETERMINATION OF CELL

NUMBER

aseptically pipette 5 ml of sterile saline solution into 6 tubes

aseptically withdraw 5 ml of the original cell suspension into the first tube (you prepare

dilution 2-1) and then homogenize with vortex mixer

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aseptically withdraw 5 ml of suspension from 2-1 dilution into the second tube

(you prepare dilution 2-2) and then homogenize with vortex mixer

aseptically withdraw 5 ml of suspension from 2-2 dilution into third tube (you

prepare dilution 2-3)

and then homogenize with vortex mixer

aseptically withdraw5 ml of suspension from 2-3 dilution into the fourth tube

(prepared by diluting your 2-4) and then homogenize with vortex mixer

aseptically withdraw 5 ml of suspension from 2-4 dilution into the fourth tube

(prepared by diluting your 2-5) and then homogenize with vortex mixer

1 tube with pure cultivation media is blank

measure the absorbance of the all dilutions against blank at 550 nm by

spectrophotometer and prepare analytical line A = f (dilution)

B) PROCEDURE OF DETERMINATION OF VIABLE CELL BY CULTIVATION

METHOD

aseptically pipette 4.5 ml of sterile saline solution into 4 tubes

aseptically withdraw 0.5 ml of the cell suspension into the first tube (you prepare

dilution 10-1) and then homogenize with vortex mixer

aseptically withdraw 0.5 ml of suspension from 10-1 dilution into the second tube

(you prepare dilution 10-2) and then homogenize with vortex mixer

aseptically withdraw 0.5 ml of suspension from 10-2 dilution into third tube (you

prepare dilution 10-3) and then homogenize with vortex mixer

aseptically withdraw 0.5 ml of suspension from 10-3 dilution into the fourth tube

(you prepare dilution your 10-4) and then homogenize with vortex mixer

aseptically withdraw 0.5 ml of suspension from 10-4 dilution into the fourth tube

(you prepare dilution 10-5) and then homogenize with vortex mixer

aseptically add 0.2 ml of suspension of all the dilutions on the surface of Petri

dishes

spread a suspension on the surface of Petri dishes with a sterile stick and

incubated in the inverted position for 48 hours at 30C

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at the end of the incubation period, select all of the Petri plates containing

between 30 and 300 colonies.

The number of yeasts per 1 ml in the original sample can be calculated as follows

( ml/CFU ):

D*5*pml/CFU (5)

ml/CFU is the total number of colony forming units per 1 ml of original sample, p is

the average number of colonies on Petri dishes in tube with a given dilution, 5 is

recalculation of the number of colony units per 1 ml, D total dilution of the sample.

C) PROCEDURE OF DIRECT COUNTING CELL IN BURKER CHAMBER

Attach the cover slide on a clean chamber Burker.

Stir thoroughly test tube with dilution of 2-5 and transfer a drop in the centre of

computing

chamber. Check that the number of yeasts on surface of a one small square

(1/25 mm 2) is in the range 8-12. If necessary, take a cell suspension with more

(2-6) or less dilution (2-4).

count least the cells in 8 squares at a magnification of 400 ×. Cells lying or

touching the borderline squares are counted only if they are on the top or right

side of the square.

Calculate the total number of cells per 1 ml (P/ml) of original suspension

according to

the formula (4)

D) PROCEDURE OF DRY CELL MASS DETRMINATION

insert empty-cellulose acetate membrane on aluminium weighing pan

dry empty membrane to constant weight at 105C

after drying marks the mass of the empty membrane shown on display

Filter the 15 ml of the cell suspension using a vacuum pump

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wash filter cake on the membrane several times with saline solution

insert wet cellulose acetate membrane with filtration cake on aluminium weighing

pan

dry wet membrane with filtration cake to constant weight at 105 °C

The concentration of yeasts dry mass per 1 ml (mg/ml) of the original sample can be

calculated as follows:

V

mmc 01 (6)

where 1m , 0m , V are weight of membrane with cell, empty membrane and volume of

filtered suspension.

LABORATORY REPORT AND QUIZ

Process the results of determining the cell number in tables:

Spectrophotometry

Dilution

20 2-1 2-2 2-3 2-4 2-5 2-6 2-7

Absorbance 2.00 1.57 0.82 0.62 0.34 0.13 0.10 0.03

Plate Cultivation Method Dilution

10-3 10-4 10-5 10-6 10-7 10-8

Number of Colonies (Plate A)

TMTC TMTC TMTC 46 5 -

Number of Colonies (Plate B)

TMTC TMTC TMTC 58 6 -

Number of Colonies (Average)

52 5.5

CFU/ml

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Burker Chamber Method

Dilution

10-1 10-2 2-5 2-5

Number of Cells per square (1/25 mm2) (Plate

A) 39 2 13 9

Number of Cells per square (1/25 mm2) (Plate

A) 37 5 11 16

Number of Cells per square (1/25 mm2)

(Average) 38 3.5 12 12.5

CFU/ml (in original sample)

Questions:

1- In excel plot the calibration line as dependence A550 = f (number of cells)

2- How can you determine the number of microbial cells in the environment?

3- Which of above mentioned methods is the fastest?

4- Which methods are suitable for determining number of living cells?

5- Why we evaluate only nutrient agar plates containing 30 -300 colonies?

6- When and how to perform gravimetric determination of dry cells?

Answers:

1- Calibration Line in Excel

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 10000000 20000000 30000000 40000000 50000000 60000000

Ab

sorb

ance

Number of Clls (Cells/ml)

Absorbance-Number of Cells

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2- By using different methods such as spectrophotometer, dilution and macroscopic

counting, and cell dry mass.

3- Spectrophotometer

4- Counting colony forming unit (CFU/ml).

5- Because if the number of colonies are less than 30, then the counting mistake

will be more and if the number of colonies are more than 300, then its

macroscopic counting would be difficult.

6- Mostly when we are working with filamentous fungi and need to measure the

quantity of its growth, we use this method. Counting filamentous fungi colonies is

a very difficult work, as they do not grow in such colonies as bacteria and yeast.

Laboratory Exercise 15

The effect of chemical agents on bacteria

Chemical agents can influence the growth of bacteria. Chemicals can have cidal or

static effect on microorganisms. Cidal effect of the chemicals can kill the

microorganisms, but chemicals with static effect can only stop growth and will not kill the

microorganisms.

It can happen that chemicals which are microbicidal, can have microbistatic effect if it is

used in low concentrations. We are using disk diffusion method for measuring the

activity of different chemicals.

THE PRINCIPLE

We know of a number of antimicrobial agents. They are used as disinfectants,

preservatives or as medicines. Some of them are produced by microorganisms such as

filamentous fungi or streptomycetes, we call them antibiotics. Others are synthesized in

the laboratory and we call them chemotherapeutics. They may have microbicidal or

microbiostatic effects. Many microorganisms are resistant to antimicrobial compounds,

either primary or secondary (due to e.g. mutations). Methods for testing the

susceptibility of microorganisms to antimicrobial compounds can be divided into two

categories.

In the disk- diffusion method a Petri plate containing an agar growth medium is

inoculated uniformly over its entire surface. Antibiotics are impregnated onto paper

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disks and then placed on a seeded agar. The plate is then incubated and the diameter

of the zone of inhibition around the disk is measured to the nearest millimetre. The

inhibition zone diameter that is produced will indicate the susceptibility or resistance of a

cell to the antibiotic. For example, a zone of a certain size indicates susceptibility, zones

of a smaller diameter or no zone at all shows that the bacterium is resistant to the

antibiotic. Size of zone depends on size of the inoculum, distribution of the inoculum,

incubation period, and depth of the agar, diffusion rate of the antibiotic, concentration of

antibiotic in the disk, and growth rate of the bacterium. This method is qualitative and

may also be used to measure the sensitivity of any microorganism to a variety of

antimicrobial agents. Diffusion tests are primarily qualitative methods that normally

should only be used to report whether a bacterium is resistant or not. Dilution method is

quantitative and involves subjecting the cell to a series of concentration of antimicrobial

agents in broth environment. The lowest concentration at which the cell is completely

inhibited (as evidenced by the absence of visible bacterial growth) is recorded as the

minimal inhibitory concentration or MIC. The MIC is thus the minimum concentration of

the antibiotic that will inhibit cells. Growth (or growth inhibition) depends on the

concentration of antimicrobial compound in the medium, so there is a dependency,

called curve toxicity.

EQUIPMENT FOR A COUPLE OF STUDENTS

Petri plate containing nutrient agar, cotton swab, antibiotic disks, tweezers, ruler, tips

and pipette

Culture: Bacillus subtilis, Escherichia coli, Micrococcus luteus, Serratia marcescens

PROCEDURE

Aseptically swab the assigned cultured onto the appropriate plate. Swab in three

directions to ensure complete plate coverage. Let stand at least 5 minutes.

Sterilize forceps by dipping in alcohol and burning off the alcohol. Obtain a disk

impregnated with a chemotherapeutic agent and place it on the surface of the

agar. Gently tap the disk to ensure better contact with the agar.

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Incubate the plate inverted until the next period. Measure the zones inhibition in

millimetres using a ruler on the underside of the plate.

LABORATORY REPORT AND QUIZ

Process the results in table below.

Antimicrobial

Agent

(size of

inhibition

zone in mm)

Bacillu

s

subtilis

Micrococcu

s luteus

Escherichi

a coli

Serratia

marcescen

s

Saccharomyc

es

cerevisiae

Candid

a utilis

Giseofulvin

(Anti-fungal) 0 0 0 0

0 0

Ampicillin

500mg (Anti-

bacterial)

2 0 2.5 3.0

0 0

Betnesol-N

Eye

Ointment

(Anti-

bacterial

0.5 0.1 1 0

0 0

Clotrimazolu

m 100mg

(Anti-Fungal)

2.5 0 2.5 0.2

0 0

Questions:

1. In which growth phase is an organism most sensitive to an antibiotic?

2. What is the difference between microbicidal and microbiostatic?

3. Why can not compare the effect of antibiotics by diffusion method?

4. Which bacteria are more sensitive to antimicrobial compounds and why?

5. Think about how resistance can develop.

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Answers:

1- In the cell division phase

2- Microbicial kills the microorganisms and microbistatic just stop their growth and

after removing the effect of the microbistatic agent, the microbe will be able to

continue its growth again.

3- Because in this method we do not use antibiotics with different concentrations to

be able to compare the antibiotics to each other.

4- Gram positive bacteria are the most sensitive bacteria against antimicrobial

agents and chemicals.

5- If we apply antibiotic to microorganisms in lower concentrations than their

microbicidal dose, a number of them will die, and some will survive. The once

who survived are much stronger than before, so it can be a method for

strengthening of useful microorganisms in industries.