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”

2

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

3

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

5

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.

6 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)

7 

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.

8 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

9 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 -1 Sample of soil, sterile saline solution, sterile tubes labelled with wax pencil as 10 , -2 -3 -4 10 10 , 10 , sterile tips, micropipette, vortex, sterile spreader, agar plates

Comment [M1]: L_shape stick or spreader

labelled with name and dilution as -1 -2 -3 -4 10 , 10 , 10 , 10 , 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 -1

prepare dilution 10 ) and then tube homogenize with vortex mixer

Comment [M2]: 0,5 g of soil

10 

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

Table of results Dilution Nutrition Agar Malt Extract Agar

Plate 1 Plate 2 Average CFU/ml Plate 1 Plate 2 Average CFU/ml

Non diluted TMTC TMTC TMTC TMTC TMTC TMTC TMTC

10-1

10-2

10-3

10-4

TMTC TMTC TMTC TMTC TMTC TMTC TMTC

TMTC TMTC TMTC TMTC TMTC TMTC TMTC

11 40 26

4 3 4

125 110 118

2 0 1

- 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 ( CFU / ml ):

CFU / ml  p * 10 * D

(3)

11

CFU / ml 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)

12 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.

13 Laboratory report and quiz Table of results Group of Cultivation Microorganisms Medium

Conditions of Cultivation

Bacteria

Nutrition Agar

Temperature: 25°C Time: 3 days Light mode: normal

Yeast

Malt Extract Agar

Temperature: 25°C Time: 3 days Light mode: normal

Count of Colony Types

26

18

Colony Description Diameter: 1mm Appearance: circular Margin: entire Elevation: flat Color: white 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…..

14 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)

15 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

16 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:

Colony Description

0.1ml at: 25°C; For: 7 days

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?

17 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

18 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

19 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.

20 LABORATORY REPORT AND QUIZ Comment [H4]: Saccharomyces cerevisiae?

Table of observations Appearance

Bacillus subtilis

Micrococcus luteus

Saccharomyces cerevisiae

Tetrads, oval shaped, and raised elevation

Oval

Brownian movement, Fluid movement

Same like m.l.

Draw the types of microorganism s observed:

Shape and arrangement:

Motility:

Rod shaped, convex elevation, and entire margin True motility, Brownian movement, Fluid movement

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.

21 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.

22

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



Let the smear air dry.

23 

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?

24 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 grampositive 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

25 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.

26 

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 Bacillus subtilis

Escherichia coli

Micrococcus luteus

Morphology, arrangement, relative size

Rod Shaped

Rod Shaped

Tetrads

Color

Dark blue

Red

Dark blue

Gram reaction

Gram Positive

Gram Negative

Gram Positive

Appearance

Sketch a few bacteria.

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.

27 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 broadspectrum 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.

28 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.

29 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.

30 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.

31 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

32 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

Saccharomyces cerevisiae Color: cream

Draw a Typical Colony

Wet Mount

33

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

34

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

35 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

36 

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 Cells ++ +++ 14 Days old Bacillus subtilis Temperature 20 40 Cells +++ +++ Escherichia coli Temperature 20 40 Cells ++ +++

50 +

60 +

70 -

80 -

90 -

100 -

50 ++

60 ++

70 +

80 +

90 -

100 -

50 +++

60 ++

70 ++

80 +

90 +

100 -

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?

37 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.

38 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 2 3 4 5 6 7 8 9 10 MO 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 0.42 2.00 2.00 1.55 1.22 0.42 0.14 0.09 0.08 cerevisiae Rhudutorula 0.79 0.07 0.68 0.49 1.05 1.22 0.81 0.79 0.73 glutinis

39 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

40 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 520%, 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.

41 

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 0% NaCl 0.5% NaCl 5% NaCl 10% NaCl 20% NaCl 25% NaCl

++++ ++++ +++ + -

S. marcescence ++++ ++++ -

B. subtilis

M. luteus

++++ ++++ +++ + + -

+++ ++ + -

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: 1234-

Bacillus subtilis Bacillus subtilis Foods with higher salt concentrations. Osmosis – the process of transferring of small molecules through semipermeable membrane and not transferring larger molecules.

42 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.

43 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

Paper

0 sec

30 sec

60 sec

90 sec

120 sec

44 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.

45 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.

46

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:

P  p * 250 000 * D

(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

47 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

48 

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

49 

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 ( CFU / ml ):

CFU / ml  p * 5 * D

(5)

CFU / ml 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

50 

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:

c

m1  m0 V

(6)

where m1 , m0 , 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: Dilution Spectrophotometry

Absorbance

20

2-1

2-2

2-3

2-4

2-5

2-6

2-7

2.00

1.57

0.82

0.62

0.34

0.13

0.10

0.03

10-6

10-7

10-8

46

5

-

58

6

-

52

5.5

Dilution Plate Cultivation Method 10-3 Number of Colonies (Plate A) Number of Colonies (Plate B) Number of Colonies (Average) CFU/ml

10-4

10-5

TMTC

TMTC TMTC

TMTC

TMTC TMTC

51

Dilution Burker Chamber Method Number of Cells per square (1/25 mm2) (Plate A) Number of Cells per square (1/25 mm2) (Plate A) Number of Cells per square (1/25 mm2) (Average) CFU/ml (in original sample) Questions: 123456-

10-1

10-2

2-5

2-5

39

2

13

9

37

5

11

16

38

3.5

12

12.5

In excel plot the calibration line as dependence A550 = f (number of cells) How can you determine the number of microbial cells in the environment? Which of above mentioned methods is the fastest? Which methods are suitable for determining number of living cells? Why we evaluate only nutrient agar plates containing 30 -300 colonies? When and how to perform gravimetric determination of dry cells?

Answers: 1- Calibration Line in Excel

Absorbance

Absorbance-Number of Cells 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0

10000000 20000000 30000000 40000000 50000000 60000000 Number of Clls (Cells/ml)

52 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

53 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.

54 

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

Saccharomyc es

Candid a utilis

0

0

0

0

0

0

0

0

Bacillu Micrococcu Escherichi s s luteus a coli subtilis

Serratia marcescen cerevisiae s

Giseofulvin (Anti-fungal)

0

0

0

0

Ampicillin 500mg (Antibacterial)

2

0

2.5

3.0

(size of inhibition zone in mm)

Betnesol-N Eye Ointment (Antibacterial

0.5

Clotrimazolu m 100mg 2.5 (Anti-Fungal)

0.1

1

0

0

2.5

0.2

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.

55 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.