MICROBIOLOGICAL PROJECT

IDENTIFICATION OF UNKNOWN BACTERIA

By Mayang Gitta Pawitra A report submitted to the University of Birmingham For the course of Bioscience for Engineers (04 18520)

School of Chemical Engineering The University of Birmingham November - 2013

ABSTRACT

Bacteria contamination can occurs in nearly every area of humans life especially in the field related to food and drink processes, for example in brewing process. Bacteria is often found in the process of brewing as the major contaminant, affecting the taste and physical appearances of beer resulted from the process Bacteria is a microscopic creature which makes it hard for human eye to observe, therefore to identify bacteria, scientist used a technique known as Gram Staining. The focus of this experiment was to identify bacteria contaminant from a given sample by looking at its physical appearances (size, shape and colour) through processes of isolating, staining and microscopic observation. From the gram-staining experiment, bacteria 10 were proven to be gram positive bacteria shown by the purple-bluish colour produced during microscopic observation. Bacteria 10 has a large rod-shaped cells, 5 in length and tends to be isolated or in a chain made of several rods joined end to end. From the observation through microscopes, bacteria 10 shows a lot of resemblance to bacillus megaterium in term of size and shape of the cell. Other experiment was then conducted to compare the growth of sample and bacillus bacteria through different media. After being incubated for 24 hours, bacteria 10 grew in a similar pattern to bacillus megaterium in all media (McConkey agar, EMB agar and CLED medium). Therefore it can be concluded that sample bacteria 10 is bacillus megaterium. Bacillus megaterium is a bacteria that grows well in soil. Although it rarely occurs in brewing but it is predicted to have infected beer due to un-hygiene process of malting. The test used throughout the experiment may have been outdated, but scientists still do it in application as their first step in order to identify an unknown bacteria. But the result would have been more precise and the bacteria would be easier to identify if more test is undergone after gram staining test. Since different bacteria have different reaction towards each test, it would be more accurate to analyze it rather than to predict it based on its physical appearances.

Keywords: Bacillus megaterium, brewing, bacteria contamination, gram staining

TABLE OF CONTENTS

Abstract................................................................................................... Table of contents.................................................................................... List of figures......................................................................................... List of tables........................................................................................... CHAPTER 1: Introduction..................................................................... CHAPTER 2: Methods and Materials.................................................... a. Materials............................................................................................. b. Method................................................................................................ b.1 Distillation method and Platting Bacteria......................................... b.2 Gram stain........................................................................................

2 3 4 5 6 7 7 7 8 9

CHAPTER 3: Result............................................................................... 10 CHAPTER 4: Discussion....................................................................... 12

CHAPTER 5: Conclusion....................................................................... 15 references................................................................................................ 16

LIST OF FIGURES

Fig 1. Equipment used during the experiment.................................. Fig 2. Serial dillution technique........................................................ Fig 3. Colour changes occurs in staining process............................. Fig 4. Microscopic pictures of sample and bacillus megaterium..... Fig 5. Bacteria 10 and bacillus megaterium in different media....... Fig 6. Flow chart gram stain............................................................. Fig 7. The brewing process...............................................................

8 9 9 10 11 12 14

LIST OF TABLES

Table 1. Staining and morphology of bacteria.................................. Table 2. Colony characters of the bacterial isolates.......................... Table 3. Experiment and its result.....................................................

10 10 13

CHAPTER 1 INTRODUCTION

The environment is filled with microorganism such as bacteria and fungi populating the air for the human to breathe in and the water for consumption. Some microorganisms are useful for human life but some others are dangerous and might cause several illness, for example bacteria contamination in brewing process. The brewing process is the process of making beer and it is known to easily get contaminated. Bacteria is often found in the process of brewing as the major contaminant, affecting the taste and physical appearances of beer resulted from the process (Storgards 2000). Type of bacteria often found in beer are bacillus, pediococcus, acetobacter, zymomonas and megasphaera (Barnes 2011). Product of contaminted brewing tends to be off-flavours, moldy, slimier and more acidic comparing to beer which are not contaminated. Bacteria contamination during brewing is likely to occur due to unsanitized equipment, in contact with unsterilled water, air, yeast culture or it could also be occured during the process of packaging. (Lowry, 2011) Nevertheless observing microorganism is a challenging task due to its microscopic form and colorless. To observe microorganism more accurately, a scientist named Hans Christian Gram developed a technique known as Gram Stain (Bergey 1994). According to David Bergey (1994), gram staining uses the principle of comparing bacterias cell wall from their chemical and physical properties as a quicker method in characterising bacteria (Bruner 1933). Gram staining characterize bacteria into Gram-positive and Gram-negative depending on their colour. The change of colour occurs during staining process would enable the bacteria to be examined by using microscope. (Kaplan 1933) Nowadays, the gram staining method is still applied in diagnosing bacteria and yeast as it used in this experiment. The focus of this experiment is to identify contaminant from a given sample by looking at its physical appearances (size, shape and colour) through processes of isolating, staining and microscopic observation.

CHAPTER 2 METHODS AND MATERIALS

A. Materials 250 ml of gram crystal violet produced by BENEX Limited, Ireland 250 ml of gram Iodine produced by BENEX Limited, Ireland 250 ml of decolorized produced by BENEX Limited, Ireland 250 ml of counterstain produced by BENEX Limited, Ireland 12 plates of Nutrient Agar (NA) and Malt Extract Agar (MEA) 500 ml of distilled water (placed in a bottle) 1 bunsen burner and a lighter A stopwatch 1 pipette manufactured by Finpipette 1 pipette manufactured by BioHit Loops Glass slide Gram stain kit Microscope (merk: Olympus, serviced by RESOLUTION) Incubator 6 test-tubes on a test tube rack Colony counter manufacture by Reichert Quebec Rotamixer manufactured by Hook and Tucker Instrument LTD, England

1. Rotamixer

2. Microscope

3. Gram stain kit

4. Pipettes

5. Distilled water

6. Colony counter

7. Bunsen burner

8. Stopwatch

9. Microscope

Fig. 1 Equipment used during the experiment

B. Method B.1 Dillusion Method and Platting Bacteria (referring to lab manual) 1. There were a set of 6 dilution tubes along with a bottle of the main sample 2. To avoid the main sample from contamination, 30 mL of the main sample were poured into another bottle called the working bottle. 3. Using a pipette, 0.1 mL of sample were taken from the working bottle and added to the first tube of dilution series and mixed by using rotamixer. 4. Another 0.1 mL were removed from the 1st tube into the 2nd tube and a mixing process occured 5. The same step were repeated for the 3rd until the 6th tube 6. 0.1 mL from the 4th, 5th and 6th tube were taken onto a Nutrient Agar and Malt Extract Agar dish. The bacteria were spreaded evenly over the suface of agar by using a sterile plastic spreader and was placed in a 35-37 C incubator for 24 hours. 7. Also platted the undiluted cell culture for comparision to the dilluted culture. 8. The bacterial colonies produced was then used for gram-staining processes.

Fig 2. Serial dilution technique (Sources. Successive Serial Dilution)

B.2 Gram Stain (referring to lab manual and Beveridge TJ, Davies JA 1983) 1. A sample was taken from isolated culture and put on a glass slide 2. 1 -2 drops of water were added onto the glass side and spreaded evenly to form a thin smear 3. The smear was then heated on top of bunsen burner allowing it to fix onto the glass 4. Crystal violet was flooded onto the glass slide for 1 minutes and washed by distilled water. 5. Iodine was added on the glass slide for 1 minutes and washed off by distilled water. 6. Smear was then decolorized by acetone for 30 second and rinsed gently by distilled water 7. Counterstain was added to the smear for 1 minutes and washed gently by water. Counterstain was applied last to stain the decolorised gram-negative bacteria a pink or red shade. 8. The smear was then analyzed by using microscope to identify the characteristic of bacteria, whether it is a gram-positive or gram-negative bacteria.

Fig 3. Colour changes occurs in staining process (Sources. Gram stain technique)

CHAPTER 3 RESULT

Morphology of Bacteria Sample Bateria 10 was observed using microscope to get a clear view of its shape, colour and its colony. Observation was done after the staining process. With the help of adding colour to bacteria, it helps analyzing the sample more accurately.
Table 1. Staining and morphology of bacteria

Isolate Sample 10 B. megaterium B. subtilis

Gram character Positive Positive Positive

Shape of cell Long rod Long rod Short rod

Arrangement of cell Long chain Long chain Long chain

Sources. Bacterial Growth Characteristics Table 2. Colony characters of the bacterial isolates

Bacteria isolate Sample 10 B. megaterium B. subtilis

Shape Round Round Round

Colour White White Dirty white

Margin Entire Entire Lobate

Opacity Opaque Opaque Opaque

Surface texture Smooth and shiny Smooth and shiny Dry

Sources. Christoper (2003)

Microscopic pictures of sample After the process of staining, sample 10 and bacillus megaterium were photographed by using Microscope Axiolab.

Sample

Bacillus Megaterium

Fig 4. Microscopic pictures of sample and Bacillus Megaterium

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Observation of sample growth on selective and differential media Differential media are employed to differentiate certain closely related organism. Depending on the presence of specific dyes or chemical in the growth media, the organism will tend to produce certain specific characteristic changes or growth patterns that can be used for further identification steps.

(a)

(b)

(c)

Fig 5. Bacteria 10 and Bacillus Megaterium in different media (a. CLED Medium b. Eosin Methylene Blue Agar c. MacConkey Agar)

Calculation of Bacteria Sample (10) concentration in sample (CFU/ml) Concentration = = 290 SD = = 197.93 Concentration of Bacteria 10 is between 9.207 106 CFU/ml brew to 48.793 x 106 CFU/ml brew. 105 CFU/ml brew

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CHAPTER 4 DISCUSSION

The identification of unknown bacteria 10 was difficult to designate because lack of adequate test done during the experiment. There are a number of possible types of bacteria and predict on what the unknown bacteria 10 really is based on its appearances. However by using gram staining, those bacteria were able to be grouped into 2 groups, gram positive and gram negative as shown in fig 6.

Gram stain
Positive: Bacillus Megatarium Bacillus Subtilis Staphylococcus Negative: Saccharomyces Pseudomonas E.coli Acetobacter morphology Rods: Bacillus megatarium Bacillus subtilis Cocci: staphylococcus morphology Rods: Pseudomonas E.Coli Cocci: Acetobacter Saccharomyces

Fig 6. Flow chart Gram stain for testing unknown bacteria

From the experiment, bacteria 10 were proven to be gram positive bacteria shown by the purple-bluish colour produced during microscopic observation. It can be seen that bacteria 10 has a large rod-shaped cells, 5 in length and tends to be isolated or in a chain made of several rods joined end to end (fig 4). The experiment was narrowed by the fact that the bacteria were rod-shaped and gram positive. There were 2 choices of bacteria under that category which are bacillus megatarium and bacillus subtillis.

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Bacillus subtilis was eliminated from the discussion because based on the experiment conducted by Goldrick Setlow (1983) bacillus subtilis has smaller size comparing to bacillus megaterium and the colonies shaped very differently through microscopes. From the observation through microscopes, bacteria 10 shows a lot of resemblance to bacillus megaterium in term of size and shape of the cell. Other experiment was then conducted to add more evidence to support the decision on the name of bacteria 10. This experiment consists of comparison between sample and bacillus bacteria through different media. During the experiment, sample 10 and bacillus bacteria were platted onto 3 different media, MacConkey Agar, Eosin Methylene Blue Agar, and CLED Medium. After being incubated for 24 hours, it can be seen in table 3 that bacteria 10 grew similarly to bacillus megaterium in all media. Both bacteria 10 and bacillus megaterium are white colour, smooth and shiny in shape of colonies. Therefore, based on the results from previous experiments that have been done, it can be concluded that bacteria 10 is bacillus megaterium.
Table 3. Experiment and its result
Test Purpose Reagents Observation Result

Gram Stain

Bacteria comparision

To determine the gram reaction of the bacteria To compare the growth of bacteria on different media

Crystal Pink rods violet, iodine, alcohol, safranin CLED media EMB Agar MacConkey agar Visually the growth of bacteria 10 looks very similar to B.megaterium

Gram negative

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According to Erna Stortgards (2000),

contamination of bacteria in the brewery are usually divided into primary contamination originating from the yeast, wort, fermentation, maturation and secondary contamination originating from bottling, canning or kegging(fig 7).

Fig 7. The brewing process

Bacillus megaterium is bacteria that occurs rarely in brewing process (Brewers Laboratory Handbook). Although sometimes it is found in areas with many oxygen because bacillus requires oxygen to grow. They are inhibited by high levels of alcohol and can grow very quick under aerobic condition (Barnes, 2011). Bacillus megaterium grows well in soil (Periago, 2005) and is predicted to infect beer due to un-hygiene process of malting. As shown in fig 7, malting is occurs early in the process of brewery. Malting is a process of getting soluble starch inside barley by allowing it to germinate (Palmer, John). The barley, in this case, might be contaminated by bacillus megaterium, therefore the spores grow throughout the process of brewery.Bacillus megaterium is considered as an indirect beer spoilage organism because it does not grow in finished beer but it causes off flavours. (Storgards, 2000) From the calculation of bacteria concentration, the bacteria 10 concentration in beer is ranged between 9.207 106 CFU/ml brew to 48.793 x 106 CFU/ml brew. There is no maximum or minimum to the standard of bacillus megaterium in beer because the precesence of b.megaterium does not effect on humans health but only causes changes in beers flavours.

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CHAPTER 4 CONCLUSION

In conclusion the identification of unknown bacteria 10 was proven bacillus megaterium. Based on gram stain, bacteria 10 was a gram-positive rod. Comparision of bacteria 10 and bacillus megaterium on different media showed significant resemblance between both bacterias in term of shape, size and the appearances of the colony. Bacillus megaterium is bacteria found in soil and it is predicted to infect beer in the process of malting. Although, it is very rare to find bacillus megaterium in beer, but the precense of this bacteria could effect to the change of flavours in the final product of beer. The test used throughout the experiment may have been outdated, but scientists still do it in application as their first step in order to identify an unknown bacteria. But the result would have been more precise and the bacteria would be easier to identify if more test is undergone after gram staining test. Those tests are Indole test, methyl red test, nitrate reduction broth test, catalase test, coagulase test, oxidase test and citrate utilization test. Since different bacteria have different reaction towards each test, it would be more accurate to analyze it rather than to predict it based on its physical appearances.

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References: Bergey, David H; John G.Holt; Noel R.Krieg; Peter H.A Sneath. 1994. Bergeys Manual of Determinative Bacteriology (9th ed). Lippincott Williams & Wilkins. Beveridge TJ, Davies JA. 1983. Cellular Responses of Bacillus Subtilis and Eschericia Coli to the Gram Stain. Journal of Bacteriology 156 (2): 846-58 Bruner DW. 1933. Differentiation between Gram-positive and Gram-negative Microrganisms by the use of Enzymes. J Bacteriol. 26(4):361371. Christopher, Kimberly and Bruno Elsa. 2003. Identification of Bacterial Species. Department of Biological Sciences. University of Alberta. Canada. Kaplan ML, Kaplan L. 1993. The Gram Stain and Differential Staining. J Bacteriol. 25(3):309321. Lowry, David: Swalweel, Malcolm. 2011. An Introduction to Beer Spoilage Organism and Flavour Taints in Beer. IBD Hygiene Symposia Pacific Region. INCOLAB Periago, Paula et all. 2005. Bacillus megaterium spore germination and growth inhibition by a treatment combining heat with natural antimicrobial. ISSN (17). 1330-9862 Setlow, Goldrick. 1983. Expression of a Bacillus Megaterium sporulation specific gene during sporulation of Bacillus subtilis. PubMed; 155(3):1459-62 Storgards, Erna. 2000. Process Hygiene Control ing Beer Production and Dispensing. VTT Biotechnology. University of Helsinki Bacterial Growth Characteristics. Available at http://www.haspi.org/curriculum-library/A-P-CoreLabs/17%20Lymphatic%20System/Labs%20&%20Activities/Lab%20-%20Bacterial%20Growth.pdf [Accessed 13/10/2013] Barnes, Thomas. 2011. Off-Flavors from Bacterial Infection. Available at http://unyha.com/documents/bjcp/Off-Flavor_Training_Part_II_-_Infections(1).pdf [Accessed 13/10/2013] Brewers Laboratory Handbook: Brewing without the blindfold. Available at http://www.brewingscience.com/PDF/BSI_brewers_lab_handbook.pdf [Accessed 14/10/2013] Palmer, John. How to brew. Available at http://www.howtobrew.com/intro.html [Accessed 14/10/2013] Successive Serial Dilutions. Available at http://biology.kenyon.edu/courses/biol09/tetrahymena/serialdilution3.htm [Accessed 16/10/2013] Whyeast Laboratories. Fermentation. Available at http://www.wyeastlab.com/he_b_fermentation.cfm [Accessed 16/10/2013] Gram Stain Technique. Virtual Amrita Laboratories Universalizing Education. Available at http://amrita.vlab.co.in/?sub=3&brch=73&sim=208&cnt=1 [Accessed 16/10/2013]

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