UNIVERSITY OF MALAWI CHANCELLOR COLLEGE BIOLOGY DEPARTMENT LABORATORY REPORT - BIO 310 BACTERIA

FOR: Mr. P.G Mpeketula

BY: Chiletso B. Phiri

REG #: BSC/11/06

DUE DATE: 26th may,2009.

TABLE OF CONTENTS

1. Abstract 2. Introduction 3. Materials and methods 4. Results 5. Discussion 6. Conclusion 7. Reference

ABSTRACT
Bacteria display many cell morphologies and arrangements. Bacteria display a wide diversity of shapes and sizes, called morphologies. Bacterial cells are about one tenth the size of eukaryotic cells and are typically 0.5 5.0 micrometres(m) in length. They also vary in their motility and flagellar arrangement. In this experiment the variations were investigated together with the reaction of the bacteria to the gram stain. This enables us to classify them into gram positve if they retained the gram stain and gram negative if they did not retain the gram stain after being washed by acetone. We were given four types of bacteria labeled A, B, C, D. The sizes of one of these were then measured using a graticule eye piece and a micrometer. The shapes, sizes, motolity and flagellar arrangement of each was observed and recorded. These samples were then stained with the gram stain and then safranine. With reference to the reaction of the bacteria to these two stains it was concluded that A was gram negative, B was gram negative, C was gram negtive and D was gram positive.

INTRODUCTION
Bacteria are unicellular prokaryotic organisms; consists of all organisms that are not members of the domain archea. Regardless of their minuteness, there is a wide variation in their size, shape and motility and nutritional needs. Bacteria size varies greatly from the smallest being 0.2m in diameter to the largest being 700m in diameter. The size of bacteria can be measured accurately under the microscope using an ocular micrometer and graticule eyepiece. An ocular micrometer is a disc with etched equidistant dimensionless lines. The distance between the lines is calibrated beforehand using a stage micrometer, an instrument which functions as a ruler for microscopic work. Bacterial shapes are equally variable with spherical ones also called cocci, rods also called bacillus, curved rods also called vibrio and filaments. The variation of bacteria does not end here, their flagella arrangement is another area of variation. By flagella arrangement three groups can be identified namely; polar flagellation, where flagella is attached to one or both ends, lophotrichous where groups flagella may arise at one end peritrechous where flagella are attached in many places around the cell surface. Another way of differentiating bacteria is by staining. Bacteria retain some stains and some dont and this can be used in differentiating them. One useful stain is the gram stain, where those bacteria that retain the stain are called gram positive while those that dont retain it are called gram negative.Gram negative retain a red color after being stained with safranin on the other hand gram negative dont retain the stain after being washed with acetone. In the gram stain, an insoluble Cristal violent- iodine complement is formed inside the cell, and this complex is extracted by alcohol from gram negative bacteria but not from gram positive bacteria. Gram positive bacteria which very thick cell walls consisting of several layers of peptidoglycan, become dehydrated by the alcohol. This causes the pores in the walls to close, preventing the insoluble crystal violet iodine complex from escaping. In gram positive bacteria, alcohol readily penetrates the lipids rich outer layer also does not prevent solvent passage; thus the crystal violet-iodine complex is easily washed away. However, the gram reaction is not directly related to cell wall chemistry. Thus, it is not the chemical constituents but the physical structure that is responsible for a gram-positive reaction. This experiment was aimed at demonstrating the various sizes and shapes of bacteria, observing the motility in bacteria and thus deduce flagella

arrangement and familiarizing students with various techniques used in staining bacteria cells. Identification of bacteria in the laboratory is particularly relevant in medicine, where the correct treatment is determined by the bacterial species causing an infection. Consequently, the need to identify human pathogens was a major impetus for the development of techniques to identify bacteria.

MATERIALS AND METHODS

To measure the size of bacteria; a stage micrometer was placed on the microscope stage and was observed under medium power objective at which point the lines were visible. The graticule eyepiece was inserted in one eye piece tube. The scale of the graticule was then superimposed with that of the stage micrometer. The number micrometers in each division of the graticule was calculated. This was repeated for 4x and 100x(oil) objective. The results were then recorded. A slide was removed from the alcohol with a pair of forceps, the excess alcohol was left to drip off and the rest was burnt off. The slide was then polished with tissue paper while holding it on the edges. The slide was then divided into two by a marker and labeled A on one side and B on the other side or C on one side and D on the other side. 2-4 loopfuls of A, B, C or D were aseptically transferred on appropriate slides respectively. The drop is then covered by a cover glass and the sides of the cover glass were sealed with Vaseline to reduce evaporation. The slide is the examined under 40x. The shape of the bacterium observed was drawn showing the typical shape of the bacteria. The length and width of five cells was measured and the average was found. Polluted pond water was then examined for various bacteria shapes, sizes and motility. A slide was removed from the alcohol jar with forceps. The excess alcohol was left to drip off and the rest was burnt off and the slide was then left to cool. The slide was then polished. 2-4 loopfuls of bacterial suspension were then aseptically transferred from the broth to the slide or if from a solid into a drop of water to emulsify. The drop of bacteria was then evenly spread to reduce clumping by slanting the slide lengthwise and letting the drop roll down. The smear was left to air-dry. The smear was fixed by passing the slide rapidly through the blue portion of the flame 3 or four times with the smear on the upper side and the slide was left to cool. For the gram stain; the dried, heat fixed smears were stained for one minute with ammonium oxalate/ crystal violet. This was then quickly washed and blot dried. The slide

was then flooded with iodine for one minute, it was then poured off and rewashed with fresh iodine for one to two minutes. The slide was then washed with water and blot dried. The slide was then decolorized with alcohol-acetone for 15-30 seconds until no more violet color was removed from the drops. The slide was then washed with water again and blot dried. The slide was then counterstained with safranine or carbol fuchsin for 30 seconds. The slide was then washed and blot dried and left to air dry. The slide was then examined under 40x and oil. After the slides were viewed the results were recorded in tabular form on size, motility, shape and flagella arrangement.This experiment was conducted to demonstrate the diversity of the various shapes, motility, size and flagella arrangement of bacteria.

RESULTS
For 10x objective; (100/205) = 1.205 For 40x objective; (30.9/100) =0.309 For 100x objective; undefined: unable to see stage micrometer. TYPE SHAPE MOTILITY FLAGELLAR ARRANGEM ENT None observed GRAM STAIN REACTION Gram stain was not retained after washing with acetone therefore they were gram negative. Gram stain was decolorized after washing with alcohol but retained the safranine stain. Gram stain was not retained after washing with acetone.

Rod shaped

Movement is directional . Flowing towards same direction. Movement is directional .

Oval

None observed

Oval shaped Rod shaped

None observed

Rod shaped

Movement is directional , zigzag. There is upward flow.

Have a single flagellum.

Retained the gram stain even after being washed by acetone thus it is gram positive

In pond water there were rod shaped, oval shaped and spherical shaped bacteria. There were also varying sizes small, long, microscopic but generally the sizes varied from small to large. The motility observed was movement in a straight line, zigzag motion and fluid motion.

DISCUSSION
The ecological significance of bacterial motility or the lack of it is that if the bacteria are motile they will be able to affect a larger area than when they are non-motile. And the ecological importance of bacterial shape is that some shapes are better suited for survival in certain conditions than others. The coccus for example is able to survive desiccation better due to the shape. Motile bacteria can move using flagella, bacterial gliding, twitching motility or changes of buoyancy. In twitching motility, bacterial use their type IV pili as a grappling hook, repeatedly extending it, anchoring it and then retracting it with remarkable force. Many bacteria (such as E. coli) have two distinct modes of movement: forward movement (swimming) and tumbling. The tumbling allows them to reorient and makes their movement a threedimensional random walk. The flagella of a unique group of bacteria, the spirochaetes, are found between two membranes in the periplasmic space. They have a distinctive helical body that twists about as it moves. Several Listeria and Shigella species move inside host cells by usurping the cytoskeleton, which is normally used to move organelles inside the cell. By promoting actin polymerization at one pole of their cells, they can form a kind of tail that pushes them through the host cell's cytoplasm. Gram-positive bacteria possess a thick cell wall containing many layers of peptidoglycan and teichoic acids. In contrast, Gram-negative bacteria have a relatively thin cell wall consisting of a few layers of peptidoglycan surrounded by a second lipid membrane containing lipopolysaccharides and lipoproteins. Gram negative retain a red color after being stained with safranin on the other hand gram negative dont retain the stain after being washed with acetone. In the gram stain, an insoluble Cristal violent- iodine complement is formed inside the cell, and this complex is extracted by alcohol from gram negative bacteria but not from gram positive bacteria. Gram positive bacteria which very thick cell walls consisting of several layers of peptidoglycan, become dehydrated by the alcohol. This causes the pores in the walls to close, preventing the insoluble crystal violet iodine complex from escaping. In gram positive bacteria, alcohol readily penetrates the lipids rich outer layer also does not prevent solvent passage; thus the crystal violet-iodine complex is easily washed away. However, the gram reaction is not directly related to cell wall chemistry. Thus, it is not the chemical constituents but the physical structure that is responsible for a gram-positive

reaction. This then accounts for the purple/blue color of the gram positive bacteria and the red/pink color of the gram negative bacteria.

CONCLUSION
Bacteria shape and size vary greatly, a number of bacterial cells were observed in pond water and different shapes and sizes some big some small were seen to be exhibited by bacteria. Some of the shapes observed were rods, ovals and spheres. In all of the other bacteria there were no flagella observed except for those in sample D. The motility was astounding there was directional, fluid and zigzag motion. According to the Gram stain reactions A, B and C were found to be gram negative because the stain was not retained after washing with acetone and D was found to be gram positive because it retained the gram stain even after being washed by acetone.

REFERENCE
1. http://en.wikipedia.org/wiki/Bacteria 2. http://www.wiley.com/10.111/j.1574-1691.2 3.Mpeketula P. G. M., Fundamentals of Microbiology: Essentials and Applications.(2008). 4.Madigan M. T. ,MartinkoJ. M , Parker J . Biology of Microorganisms. 8th edition(1997), Prentice-Hall.