LABORATORY REPORT

BIOCHEMISTRY AND MICROBIOLOGY

(BFT 1014)

MEMBERS AND MATRIC NO. (1)DONNY LIANG KHAI CHIUN B11A056

(2)KHOR YEE HENG B11A135

(3)LOOI KAH SHIEN B11A165

(4)YIP HON LEAN B11A438

(5) YONG WOON SING B11A442

(6) LAU SU HUI B11A565

GROUP SBP 1DATE OF EXPERIMENT 8th MAY 2012TITLE OF EXPERIMENT Experiment 7: STAINING TECHNIQUES IN

MICROBIOLOGYExperiment 8: MEDIA PREPARATION

(DEMONSTATION)Experiment 9: ISOLATION OF SOIL

BACTERIA: VIABLE TITER AND PURE CULTURE

DEMONSTRATOR (1) DR. NOOR AZLINA BINTI IBRAHIM(2) CIK WAN NORQAYYUM NADIA WAN

ARIFIN

Experiment 7: STAINING TECHNIQUES IN MICROBIOLOGYExperiment 8: MEDIA PREPARATION (DEMONSTATION)Experiment 9: ISOLATION OF SOIL BACTERIA: VIABLE TITER AND PURE

CULTURE

Introduction:

The dominant objective of these experiments is to identify the different types of bacteria and to estimate the number of bacteria present in the sample. Besides that, we are able to learn how to prepare nutritious media. We also learn the method to obtain viable titer and the method of streaking on agar plate to obtain a pure culture. In addition, we learn the techniques of heat-fixed smear and the technique of gram staining and endospore staining.

A growth medium or culture medium is a liquid or gel designed to support the growth of microorganism.1 The media have 3 physical forms namely liquid or broth media, semisolid media and solid media. The media we used in this experiment is Nutrient Agar, a solid media. The solid media are used for the surface growth of microorganism in order to observe colony appearance, pure culture isolation, storage of cultures and to observe the biochemical reactions. While in liquefied state, solid media can be poured into either a test tube or Petri Plate. Petri Plate is used in our experiments so the plate is called an agar plate.

The process after the preparation of nutrient agars is inoculation, the process of introducing microbes into the nutrient agar. There are millions of bacteria can be found in a single drop of water. Therefore, bacteria suspension is diluted serially so as to reduce the bacteria population before transfer to the nutrient agar. There are 2 methods of inoculation, the pour plate method and the spread plate method. The technique we used is spread plate method.

Growth phases are displayed in different ways in colonies growing on a solid medium. Typically, a cell divides exponentially; forming a small colony-all the descendants of the original cell. Bacterial growth is measured by estimating the number of cells that have arisen by binary fission during a growth phase. Growth can be measured by serial dilution, in which successive 1:10 dilutions of a liquid culture of bacteria are made and transferred onto an agar plate; the colonies that arise are counted.[2 ] Each colony represents 1 live cell from the original sample.

The viable titer is determined by counting colonies (CFU’s) per volume plated and multiplying by the

dilution factor. The method only counts living cells as dead cells do not reproduce to form colonies. The

viable titer is determined only from countable plates (yield of at least 30 and less than 300 colonies.)

Viable Titer= [number of colonies (CFU )

volume plated] x dilution factor

Identification of the pure isolates includes direct microscopic examination, study of cultural characteristics as well as physiological and biochemical properties of the isolate. We are going to identify the pure isolate by using 2 tests namely the Gram Stain and Schaeffer-Fulton spore Stain (Endospore Stain) in the experiment.

Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biology and medicine to highlight the structures in biological tissues for viewing and also to view organelles within individual cells. Grams Stain is used to classify the bacteria broadly. It is based on the composition of their cell walls. In Gram staining, bacteria cells take up crystal violet. Iodine is then added; it acts as a mordant, a chemical that helps retain the stain in certain cells. Those structures that cannot retain crystal violet are decolorized with 95% ethanol or an ethanol-acetone solution, rinsed, and subsequently stained (counterstained) with safranin. 4 groups of organisms can be distinguished with Gram stain namely the Gram-positive organisms, Gram-negative organisms, Gram-nonreactive organisms and Gram-variable organisms. The difference between Gram-positive and Gram-negative organisms reveals a fundamental difference in the nature of the cell walls of bacteria. Furthermore, the reactions of bacteria to the Gram stain have helped in distinguishing Gram-positive, Gram-negative, Gram-nonreactive groups that belong to radically different taxonomic groups.[3 ]

A few types of bacteria produce resistant cells called endospores. Endospore walls are very resistant to penetration of ordinary stains. When a simple stain is used, the spore will be seen as clear, glassy, easily recognizable areas within the bacterial cell. The differential Schaeffer-Fulton spore stain makes spores easier to visualize. Heat-fixed smears are covered with malachite green and then gently heated until they steam. Approximately 5 minutes of such steaming causes the endospore walls to become more permeable to the dye. The slide is then rinsed with water for 30 seconds to remove the green dye of the cell except for the endospores, which retain it. Then a counter stain safranin is placed on the slide to stain the non-spore-forming, or vegetative, areas of the cells. Cells of cultures without endospore appear red; those with endospores appear have green spores and red vegetative cells.[3 ]

References:

1 = http://en.wikipedia.org/

2 = Jacquely G.Black, 6th edition, Microbiology Principles and Explorations (page 145 to 146)

3 = Jacquely G.Black, 6th edition, Microbiology Principles and Explorations (page 68 to 71)

APPARATUS AND MATERIALS

STEP (1) : MEDIA PREPARATION (DEMONSTRATION)

Apparatus

1) Petri plates

2) Conical flask

3) Glove

4) Bunsen burner

5) Cotton wool

6) Aluminium foil

Materials

1) Distilled water

2) Dehydrated Nutrient agar (Na,Nacl)

STEP (2): ISOLATION OF SOIL BACTERIA (VIABLE TITER AND PURE CULTURE)

Apparatus

1) 8 sterile petri plates

2) Bunsen burner

3) Inoculating loops

4) Test tube

5) Balances and weighing boats

6) 6 sterile 1000 µl miropipettes

7) 4×9ml sterile dilution blanks in capped tubes and one 100ml sterile dilution blank in a bottle

8) Nutrient agar plate (day two)

Materials

1) Molten (450c) nutrient agar

2) Molten 80ml nutrient agar-7.5% NaCl

STEP (3) : STAINING TECHNIQUES IN MICROBIOLOGY

Apparatus

1) Inoculating loops

2) Slides

3) Bunsen burner

4) Tripod Stand

5) Beaker

6) Glove

7) Dropper

Materials

1) Slant Cultures (a few type of bacteria)

2)Gram staining reagents in dropping bottles (crystal violet,safranin,malachite green,saline,acetone)

PROCEDURE

STEP (1) : MEDIA PREPARATION

Two type of nutrients agar solution were prepared by using sodium and sodium chloride ( 7.5 %

of NaCl )solutions .

In the laminar flow and with the aid of the Bunsen burner , both of the nutrients solution were

poured into 5 different petri plates and labeled . The petri dishes that contained sodium solution

is labeled as NA whereas the petri dishes that contained nutrient agar solution with sodium

chloride is marked as NA+ .

The prepared petri plates are then allowed to remain undisturbed until it has cooled and hardened

.

STEP (2) : ISOLATION OF SOIL BACTERIA (VIABLE TITER AND PURE CULTURE)

Part 1 of Viable Titer

The sterile dilution blanks were marked in the following order : the 100 ml dilution blank is 10-2

and for the 4 test tubes were marked accordingly in 10-3 , 10-4 , 10-5 , 10-6

1 g of soil sample were weighted and is added into the 10-2 dilution blank and are shaken

vigorously for at least 1 minute .

1 ml solution from the 10-2 dilution blank is transferred to the 10-3 tube aseptically . The solution

is then mixed thoroughly .

By using a fresh, sterile pipette for the following succeeding step, 1ml from the 10-3 dilution to

the 10-4dilution blank and same step is repeated for the following blank.

1 ml of solution from 10-3 is transferred into the nutrient agar medium that contained

sodium(NA) and nutrient agar medium that contained sodium chloride(NA+) . Then it is drawn

into spiral manner across the medium by using glass rod.

The above procedure was repeated subsequently for the dilution blanks with dilution factors of

10-4 , 10-5 , 10-6

The petri dishes is inverted and placed in the incubator or at room temperature.

Part 2 of Viable Titer

The petri plates were remove from the incubator and the number of colonies on each plate was

count which begin with those of the highest dilution.

The result of the number of colonies, morphology of each colony, size,shape, colour of the colonies is

recorded.

STEP (3) : STAINING TECHNIQUES IN MICROBIOLOGY

Endospore Staining ( Bacillus sp )

1 drop of saline is added on the clean slide.

The loop is heated and a little sample is taken out and smear on the slide.

The slide is placed above the boiling water bath , which will serve as steam source .

Malachite green is added on the slide is allowed to cool and wait for 5 minute.

Then it is rinsed with water carefully and Safranin is added onto the slide,

followed by gentle stream of water from the faucet.

The excess water is shaken off and it is allowed to dry.

The slide is put under microscope and the result is observed.

Gram Staining ( Unknown bacteria )

1 drop of saline is added on the clean slide.

The prepared bacteria is smeared.

The slide is heated and the smear is stained with crystal violet for 1 minute.

The slide is rinsed with tap water.

Then , continue by smeared with Gram’s Iodine for 1 minute .

Then, it is rinsed with acetone until the blue colour stops coming out of the smear.

After that , it is rinsed under tap water .

Counter stained the slide with Safranin for at least 10 second.

The slide is rinsed and is let go completely dried. The slide is then placed

under microscope after all .

Pure Culture

One colony that appeared of only one type of cell is to be composed.

The inoculating loop is flamed and is allowed to cool.

After that, the surface of the colony is touched gently by using the loop.

This cell is streaked as a primary streak on the surface of a Nutrient Agar Plate,

but not more than ¼ of the plate .

The loop is re-flamed. It is allowed to cool and then streaked across the primary streak once or twice and

then continue to streak the secondary streak. It goes same to the third and fourth.

The only different things was the fourth streak and the primary streak were not joined up .

The plate is then inverted and incubated under room temperature.

The result is observed on the next day of this experiment.

Pure Culture Gram Staining for unknown bacteria

Endospore staining for Bacillus sp

RESULTS

(A)Viable Titer

Nutrient Agar (Na)

Concentration Number of Colony MorphologySize Colour Shape

10-3 TNTC

+ Creamy White

Punctiform

++ Creamy White

Punctiform

+++ Creamy White

Circular

++++ Creamy White

Irregular

+++++ Creamy White

Irregular

++++++ Creamy White

Irregular

+++++++ Creamy White

Irregular

10-4 TNTC

+ Creamy White

Punctiform

++ Creamy White

Circular

+++ Creamy White

Circular

++++ Creamy White

Irregular

+++++ Creamy White

Irregular

++++++ Creamy White

Irregular

10-5 68

+ Creamy White

Punctiform

++ Creamy White

Circular

+++ Creamy White

Circular

++++ Creamy White

Irregular

+++++ Creamy White

Irregular

10-6 13

+ Creamy White

Punctiform

++ Creamy White

Circular

Nutrient Agar + 7.5% NaCl (Na + NaCl)

Concentration Number of Colony MorphologySize Colour Shape

10-3 5

+ Creamy White

Circular

++ Creamy White

Circular

+++ Creamy White

Circular

10-4 2

+ Creamy White

Circular

++ Creamy White

Circular

10-5 Nil - - -

10-6 Nil - - -

Viable Titer= [number of colonies (CFU )

volume plated] x dilution factor

Nutrient Agar (Na)

Concentration 10-3 10-4 10-5 10-6

Viable Titer - -

681

x105

= 6.8 x 106 CFU/ml

131

x106

= 1.3 x 107 CFU/ml

Nutrient Agar + 7.5% NaCl (Na + NaCl)

Concentration 10-3 10-4 10-5 10-6

Viable Titer

51

x103

= 5.0 x 103 CFU/ml

21

x104

= 2.0 x 104 CFU/ml- -

(B)Gram Staining Unknown Sample A

Characteristic:

- Colour: Stained red- Shape: Bacillus (Rod shape)- Structure: Peritrichous (Flagella present all over the boy

surface.)

Unknown Sample B Characteristics:

- Colour: Stained Red- Shape: Vibrio- Structure: Lophotrichous (Tuft or flagella at only one end.)

Unknown Sample C Characteristics:

- Colour: Stained Purple- Shape: Coccus (Round shape)- Structure: Atrichous (Flagella absent)

(C) Endospore StainingCharacteristics:

- Colour: Stained green (Endospore), Stained red (Vegetative cell)

- Shape: Streptobacillus- Structure: Atrichous (Flagella absent)

(D) Pure Culture

DISCUSSION

In nature, many species of bacteria and other microorganism are found growing together in oceans, lakes, soil and dead organic matter. Just like human, bacteria need nutrient to grow. In order to grow

bacteria in laboratory, we need to provide all those conditions required for growth by bacteria in mature. Component of the media is nutrients (carbohydrates, serum, whole blood, and ascetic fluid, yeast extract, peptone, beef extract) solidifying agent (agar), water (tap water with low mineral content, gas distilled water or demineralised water) and additives/supplements (dyes, vitamins, amino acids, growth factors and antibiotics). After the Nutrient agar media is prepared, we can transfer the sample to the media. To prevent contamination occurred in the Nutrient agar media, we need to always autoclave used or contaminated media containing any kind of growth before discarding it. Besides, there are some precautions we can take to reduce the risk of the nutrient agar being contaminated. [1] For example:

Never add all the agar powder at once. Sprinkle agar powder slowly on the surface of hot water and stir continuously till all the powder gets dispersed. Adding too much together will result in formation of lumps.

Never use the medium immediately after autoclaving. Incubate the medium after autoclaving at 30oC for at least 3 days before using. (This rules out contamination due to improper sterilization.)

It is better to autoclave the medium in smaller flasks. This way, you can use the medium as and when required without exposing the complete medium to atmosphere.

Any change in colour, consistency or growth in medium after incubation means some kind of contamination.

Colonial morphology (The features of the bacteria that can be observe through naked eye. For example, colour, shape, edge and elevation) and Cellular morphology (difference of the individual cells that is seen under the microscope) are sometimes useful in the identification of bacteria. [2] Size, shape and arrangement of cells are often the first clue in identification of bacteria. However, since there are many “look-alikes”, method other than microscopy must be used to determine the genus and species of an organism. Bacteria populations grow extremely fast when they supplied with the nutrients and environmental condition that allow them to thrive. Through this growth, different types of bacteria will sometimes produce colonies that are distinctive in appearance. Some colonies may be coloured, some are circular in shape while others are irregular. [4]

Based on the results obtained, the growth of bacteria is numerous in nutrient agar (Na) compare to nutrient agar + 7.5% of NaCl (Na + NaCl). This is due to NaCl inhibits the growth of any bacteria either on the surface of the agar or in the agar. Presence of NaCl create a hypertonic solutions, hence high osmotic pressure removes water from cell, causing shrinkage of cell membrane. However, there have a type of bacteria known as halophile, such as Tetragenococcus halophilus which required moderate or large quantities of salt to growth. [5] This is the reason why there are less colonies present in Nutrient agar with sodium chloride.

Pure cultures contain only one kind of cells, which are assumed to be the progeny of a single cell. Such cultures are free from contamination by any other kind of living form. Thus, they are clones from

each other. In order to develop pure cultures, microorganisms are separated from mixed population. Pure culture of this organism is then developed to get similar cells. Once the organism has been isolated, it is cultured. The complete process is known as Isolation, purification and cultivation.

In order to ensure the pure culture only consist of one type of organism, we test the colonies with gram staining and endospore staining. The thickness of Gram positive cell wall and presence of more lipids in Gram negative cell walls have been more acceptable reasons for Gram Stain reactions. It is believed that the positively charged crystal violet pass through the cell wall and cell membrane and binds to negatively charged components inside the cell. Addition of negatively charged iodine binds to the positively charged dye and forms a large dye-iodine complex within the cell. Crystal violet interacts with aqueous KI-I2 via a simple anion exchange to produce a chemical precipitate. The small chloride anion is replaced by the bulkier iodide, and the complex thus formed becomes insoluble in water. During decolorization, alcohol dissolves the lipid present in the outer membrane of Gram negative bacteria and it leaches the dye-iodine complex out of the cell. A thin layer of peptidoglycan does not offer much resistance either. The dye iodine complexes are washed from the Gram negative cell along with the outer membrane. Hence, Gram negative cells readily get decolorized. On the other hand Gram positive cells become dehydrated from the ethanol treatment, closing the pores as the cell wall shrinks during dehydration. The dye-iodine complex gets trapped inside the thick peptidoglycan layer and does not get decolorized.[3]

Endospores are dormant of metabolically inactive form of a bacterium that allows it to survive the harsh environmental conditions. Spores are resistant to heat, UV radiation and chemicals because they are comprised of a tough proteinaceous covering called keratin. Endospore staining enable us to distinguish between the vegetative cells and the endospores. Because endospores have a keratin covering and resist staining, the malachite green will be forced into the endospores by heating. In this technique heating acts as a mordant. Water is used to decolorize the cells; as the endospores are resistant to staining, the endospores are equally resistant to de-staining and will retain the primary dye while the vegetative cells will lose the stain. The addition of a conterstain (safranin) is used to stain the decolorized vegetative celss. When visualized under microscopy the cells should have the following characteristics: the vegetative cells should appear pink, the vegetative cells contain endospores should stain pink while the spores should be seen as grenn ellipses within the cells. [3]

QUESTIONS

1. State a good reason for cooling the melted agar to 45°c before pouring into the Petri plates.

We do so in order to minimize to amount of steam condensation on the Petri plates lids after the agar has been poured. This can helps to minimize the contamination of the plate.

2. Are all the bacteria in soil counted by this procedure? Is this an overestimate or underestimate of the actual number of viable bacteria in soil?

All the bacteria that counted is underestimate of the actual number of viable bacteria in soil because upon incubation each viable cell will give rise to a colony either in the agar or on the agar surface. It is possible that a colony could have arisen from two or more cells that stuck together.

3. What are possible reasons that CFU underestimate the number of bacteria in soil?

First, a colony may have consisted of more than one cell that stick to one another due to the fact that not all the organisms have been separated during the dilution of soil sample. Besides, the assumption that there is one colony per organism may be false if the soil and water is not mixed properly. There is also a possibility that some bacteria were not able to grow on the nutrient agar during the culturing process of bacteria since some of them died.

4. What is a pure culture? How do you prove a culture is pure?

A pure culture is defined as the progeny form of one cell. We can prove pure culture by showing that all the colonies on the restreak are identical and Gram staining these to demonstrate all the cells in the resulting colonies are identical and the same as those on the original plate.

5. Why is it important of obtaining pure cultures?

Pure culture is important because a pure culture maybe isolated by the use of special media with specific chemical that allow the enrichment or selection of one bacteria over another. Besides, pure cultures enables the study of morphology and physiology of individual of bacteria species, their biochemical behavior and their response toward different chemicals such as antibiotics.

6. If your organisms were all blue with pink spots, what is the Gram reaction? If your organisms were all pink but had some blue spots, what is the Gram reaction?

The organisms were all blue with pink spots is showing Gram positive reaction while organisms were all pink but had some blue spots showing Gram negative reaction.

7. How can your answer to question 6 to be proven?

Success of the gram stain relies upon the integrity of the cell wall. Gram positive bacteria that have been overly heat fixed resulting in destruction of all or parts of their cell wall can appear to be pink or have pink areas (Gram negative).

8. Suppose you observed some refractile clear spots in cells that did not stain, what are two likely causes of that observation?

The two likely causes of that observation is the glass slide that potentially pathogenic bacteria are smeared on it is not dry enough and over rinsed occurred on the glass slide that washed off the bacteria.

9. What would be the reason(s) for not finding any organisms on the slide?

The reason for not finding any organisms on the slide is we over rinsed the slide when rinsed with acetone till the bacteria is being rinsed off.

10. What would you expect from a Gram stain done on a slide that was heated too HOT during the heat-fixed smear?

If for Gram positive bacteria that have been overly heat during the heat- fixed smear will results in destruction of all or parts of their cell wall can appear to be pink or have pink areas; while for Gram negative bacteria, it will still show all pink area since the cell wall of it is not as thick as Gram positive bacteria.

CONCLUSION

A standard plate count can be used to estimate the number of microorganism living in a sample. A colony forming unit (CFU) may originate form more than one cell as the concentrationof the dilution medium decreases, the number of microorganism present decreases. A viable titer is used to estimate the number of microorganism in the sample. A pure culture is the new cultures cultured from a mixed culture by method: that separate the individual cells so that when they multiply, each of them will form an individual distinct colony which consists of only one type of organism. Finally, gram staining and endospore staining will help to determine the morphology of bacteria in order to identify the genus and species of certain bacteria. REFERENCES

Books1. Kanika Sharma, Department of Botany, M.L. Sukhadia University, Udaipur, Rajasthan, India (2007) Manual of Microbiology Tools & Techniques.2. Jacquelyn G. Black (2008) Microbiology. Seventh Edition. John Wiley & Sons (Asia) Pte. Ltd.

Website3. en.wikipedia.org/wiki/Gram_staining (10 May 2012)4. www.helium.com/items/1121016-bacterial -shape-cell-arrangement-colony-morphology (11 May 2012)5. www.ehow.com/facts_5924409_effects-salt-concentration-bacterial-growth.html (11 May 2012)