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To which negatively charged components of the cell envelopes do the crystal violet complexes bind in gram staining?

To which negatively charged components of the cell envelopes do the crystal violet complexes bind in gram staining?


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The gram positive have negative components in the peptidoglycan layer in the form of teichoic acid phosphodiester bonds, and the gram negative have negative components in their outer membrane in the form of lipopolysaccharide. Is that where the crystal violet binds?


The gram staining relies on the differences in the bacterial membranes. The gram positive bacteria have a thick peptidoglycan layer on top of the cell membrane, the gram negative bacteria have a lipid layer on the outside followed by a thin peptidoglycan layer and then the cell membrane. See the image for clarification (from here):

Crytall violet dissociates into a positively charged dye molecule (CV+) and chloride (Cl-). The CV+ travels between the membrane layers (outer and inner membrane) and is trapped there by the addition of iodine. This is used to form large crytal violet-iodine complexes which are retained during the destaining process.

In the destaining process, the peptidoglycan layer of gram positive bacteria is dehydrated and therefore shrinks which prevents the staining from being washed out. In the gram negative bacteria, the outer lipid layer is removed and the peptidoglycan layer (which is much thinner) exposed which allows the removal of the stain. The teichoic acid is important for the rigidity of the membrane but not for the binding itself.

References:

  1. Gram staining
  2. The Gram Stain after More than a Century
  3. Use of the Gram stain in microbiology

Bacterial cell envelopes fall into two major categories: a gram-positive type and a gram-negative type, distinguished by Gram staining. Either type may have an enclosing capsule of polysaccharides for extra protection. As a group these are known as polysaccharide encapsulated bacteria.

As in other organisms, the bacterial cell wall provides structural integrity to the cell. In prokaryotes, the primary function of the cell wall is to protect the cell from internal turgor pressure caused by the much higher concentrations of proteins and other molecules inside the cell compared to its external environment. The bacterial cell wall differs from that of all other organisms by the presence of peptidoglycan (poly-N-acetylglucosamine and N-acetylmuramic acid), which is located immediately outside of the cytoplasmic membrane. Peptidoglycan is responsible for the rigidity of the bacterial cell wall and for the determination of cell shape. It is relatively porous and is not considered to be a permeability barrier for small substrates. While all bacterial cell walls (with a few exceptions e.g. intracellular parasites such as Mycoplasma ) contain peptidoglycan, not all cell walls have the same overall structures. This is notably expressed through the classification into gram positive and gram negative bacteria.


STREPTOCOCCUS | Introduction

Pyogenic and Other Streptococci

The medium colistin crystal violet sulfamethoxazole trimethoprim (CCSXT) agar ( Table 8 ) is widely used for detecting group A streptococci together with some other confirmatory assays. Biochemical tests, easily performed in the laboratory, are an acceptable alternative to serological studies to identify pyogenic streptococci ( Table 9 ).

Table 8 . Composition of colistin crystal violet sulfamethoxazole trimethoprim (CCSXT) agar

Pancreatic digest of casein, powder14.5 g
Papaic digest of soybean meal, powder5 g
Sodium chloride5 g
Crystal violet0.2 mg
Colistin sulfate10 mg
Sulfamethoxazole24 mg
Trimethoprim1.25 g
Agar15 g
Distilled water950 ml

Soak for 15 min, check and if necessary adjust pH to 7.3 ± 0.2, bring to the boil to dissolve the ingredients and sterilize for 20 min at 121 °C. Cool rapidly to approximately 50 °C, add 50 ml defibrinated sheep blood, mix with gentle rotation, and pour into Petri dishes.

Table 9 . Differentiation of streptococci with the use of biochemical tests

Susceptibility toPYRCAMP testHydrolysis of hippurateBile aesculinGrowth in 6.5% NaClOptochin and bile susceptibility
BacitracinSXT
S. pyogenes (group A)++
S. agalactiae (group B)a+++ a
Large colony (group C and G)a+
S. pneumoniae +
S. equi subsp. equi d+

Symbols: +, positive −, negative d, strains dependent SXT, sulfamethoxazole and trimethoprim PYR, pyrrolidonyl arylamidase CAMP test, test for enhancement of hemolysis by Staphylococcus aureus beta lysin.

a Exceptions occur occasionally.

Lancefield group D streptococci will grow on media containing bile and may be differentiated from other streptococci by rapid hydrolysis of aesculin in the presence of 40% bile. They may be determined by using the kanamycin aesculin agar (KAA) medium ( Table 10 ).

Table 10 . Composition of kanamycin aesculin (KAA) agar

Tryptone20 g
Yeast extract powder5 g
Kanamycin sulfate0.02 g
Sodium chloride5 g
Sodium citrate1 g
Aesculin1 g
Ferric ammonium citrate0.5 g
Sodium azide0.15 g
Agar15 g
Distilled water1 l

Soak for 15 min, check and if necessary adjust pH to 7.0 ± 0.1 and bring to the boil to dissolve the ingredients completely. Sterilize for 15 min at 121 °C cool to approximately 47 °C.


Gram Stain

Figure 5. Bacteria stained with Gram stain.

In 1884, physician Hans Christian Gram was studying the etiology (cause) of respiratory diseases such as pneumonia. He developed a staining procedure that allowed him to identify a bacterium in lung tissue taken from deceased patients as the etiologic agent of a fatal type of pneumonia. Although it did little in the way of treatment for the disease, the Gram stain method made it much easier to diagnose the cause of a person’s death at autopsy. Today we use Gram’s staining techniques to aid in the identification of bacteria, beginning with a preliminary classification into one of two groups: Gram positive or Gram negative.

The differential nature of the Gram stain is based on the ability of some bacterial cells to retain a primary stain (crystal violet) by resisting a decolorization process. Gram staining involves four steps. First cells are stained with crystal violet, followed by the addition of a setting agent for the stain (iodine). Then alcohol is applied, which selectively removes the stain from only the Gram negative cells. Finally, a secondary stain, safranin, is added, which counterstains the decolorized cells pink.

Although Gram didn’t know it at the time, the main difference between these two types of bacterial cells is their cell walls. Gram negative cell walls have an outer membrane (also called the envelope) that dissolves during the alcohol wash. This permits the crystal violet dye to escape. Only the decolorized cells take up the pink dye safranin, which explains the difference in color between the two types of cells. At the conclusion of the Gram stain procedure, Gram positive cells appear purple, and Gram negative cells appear pink.

When you interpret a Gram stained smear, you should also describe the morphology (shape) of the cells, and their arrangement. In Figure 5, there are two distinct types of bacteria, distinguishable by Gram stain reaction, and also by their shape and arrangement. Below, describe these characteristics for both bacteria:

Gram positive bacterium: Gram negative bacterium:
Morphology
Arrangement

Procedure of Gram Staining

Smear Preparation

Fix material on a slide with methanol or heat. If the slide is heat fixed, allow it to cool to the touch before applying the stain.

Gram Staining Procedure/Protocol:

  1. Flood air-dried, heat-fixed smear of cells for 1 minute with crystal violet staining reagent. Please note that the quality of the smear (too heavy or too light cell concentration) will affect the Gram Stain results.
  2. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
  3. Flood slide with the mordant: Gram’s iodine. Wait 1 minute.
  4. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
  5. Flood slide with decolorizing agent (Acetone-alcohol decolorizer). Wait 10-15 seconds or add drop by drop to slide until decolorizing agent running from the slide runs clear.
  6. Flood slide with a counterstain, safranin. Wait 30 seconds to 1 minute.
  7. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then blot dry with absorbent paper.
  8. Observe the results of the staining procedure under oil immersion (100x) using a Bright field microscope.

If you are struggling to remember the staining reagents used in this procedure and their order you can remember this sentence “Come In And Stain” i.e. the order is Crystal violet, Iodine, Alcohol/Acetone and the final one is Safranin.


Difference in structure of Gram positive vs Gram negative bacteria

The diagram below illustrates the differences in the structure of Gram positive and Gram negative bacteria. The two key features that lead to the differing visualization properties of Gram positive and Gram negative species are the thickness of the peptidoglycan layer and presence or absence of the outer lipid membrane. This is because the wall structure affects the cell’s ability to retain the crystal violet stain used in the Gram staining procedure which can then be visualized under a light microscope.

Gram positive bacteria have a thick peptidoglycan layer and no outer lipid membrane whilst Gram negative bacteria have a thin peptidoglycan layer and have an outer lipid membrane.

As Gram positive bacteria lack an outer lipid membrane, when correctly referring to their structure rather than staining properties, are termed monoderms. The outer lipid membrane possessed by Gram negative bacteria means that, when referring to their physical structure, they are termed diderms.

The Gram staining technique was developed in 1884 by Danish bacteriologist Hans Christian Gram 1 . Whilst a Gram stain will not tell you the specific species you are looking at, it can be a quick way to narrow down greatly the list of potential candidates and direct follow-up testing where necessary.


GRAM-NEGATIVE VS GRAM-POSITIVE BACTERIA

  1. Cell envelope: the very first difference between Gram-positive and Gram-negative bacteria lies in the thickness of the cell wall. The cell envelope of gram-positive organisms consists of a thicker cell wall as compared to Gram-positive bacteria.
  2. Lipid membrane: Gram-positive bacteria have no outer lipid membrane whereas Gram-negative bacteria have an outer lipid membrane.
  3. Periplasmic space: the periplasm is an intermembrane structure, lying between the cell membrane and the outer membrane(unique to gram-negative cells). This space is present in Gram-negative bacteria while sometimes absent in Gram-positive bacteria.
  4. Peptidoglycan layer: In Gram-positive bacteria, the peptidoglycan layer of the cell wall is thick. However, it is thinner in Gram-negative bacteria.
  5. Resistance to osmotic pressure: because of the thicker peptidoglycan layer, the walls of gram-positive cells are more resistant to osmotic pressure than those of gram-negative bacteria.
  6. Staining dye used: crystal violet dye(purple or blue) is used to identify Gram-positive bacteria. On the other hand, safranin is used to identify the Gram-negative bacteria which gives it a pink or red colour.
  7. Resistance to antibiotics: Gram-negative bacteria are more resistant to antibiotics as compared to Gram-negative bacteria. This resistance ability of Gram-negative bacteria makes them more dangerous.

Gram Negative Cell Wall:

Gram-negative bacteria have a thinner layer of peptidoglycan (10% of the cell wall) and lose the crystal violet-iodine complex during decolorization with the alcohol rinse, but retain the counter stain Safranin, thus appearing reddish or pink. They also have an additional outer membrane which contains lipids, which is separated from the cell wall by means of periplasmic space.

Medical Relevance of Gram Negative Cell Wall:

The cell wall of Gram-negative bacteria is often a virulence factor that enables pathogenic bacteria to cause disease. The virulence of Gram-negative bacteria is often associated with certain components of the cell wall, in particular, the lipopolysaccharide ( otherwise known as LPS or endotoxin). In humans, LPS elicits an innate immune response characterized by cytokine production and activation of immune system. Inflammation occurs as a result of cytokine production, which can also produce host toxicity.


Is basic dye a negative stain?

There are many different staining techniques. Methylene blue is a simple stain that colors cells blue. In a negative staining technique, a negatively charged stain colors the background, leaving the cells light colored and unstained. The bright cells are easily visible against the dark background.

Likewise, why is applying Nigrosin as the dye considered a negative stain? It can also be used to stain cells that are too delicate to be heat-fixed. We use nigrosin as our negative stain. This means that the stain readily gives up a hydrogen ion and becomes negatively charged. Since the surface of most bacterial cells is negatively charged, the cell surface repels the stain.

One may also ask, what dye is used in negative staining?

How can a basic dye stain bacteria?

Because cells typically have negatively charged cell walls, the positive chromophores in basic dyes tend to stick to the cell walls, making them positive stains. On the other hand, the negatively charged chromophores in acidic dyes are repelled by negatively charged cell walls, making them negative stains.


Lab Report about Simple Staining of Microbes

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1.0 Introduction

Microbiology is the branch of biology that deals with microorganisms and their effect on other living organisms. Microbes are very small organisms which can only be viewed with the aid of microscope. Several groups of organisms that fit into this category are bacteria, cyanobacteria, fungi and protists. Within this group there are several species interesting to humans because of their ability to cause disease or their use in the food industry and microorganisms can be classified to unicellular and multicellular. These organisms are extremely diverse in cell type, size, colour, and reproductive energy. Microbes can be classified by their cell type. All cells can be categorized either prokaryotic or eukaryotic and the primary difference between these two cell types is the presence of a membrane-bound nucleus.

The Term Paper on Lab Report Of The Experiment Of Conjugation Of E. Coli

Conjugation is a natural occurring process that involves the transfer of DNA from one cell into another through a physical connection between the cells. In the following experiment, two strains of Escherichia coli bacterial cells (donor F’lac+strs and recipient F-lac-strr) underwent conjugation to produce a transconjugant strain (F’lac+strr). MAC plates and streptomycin were utilized .

The experiment was carried out to use a bright field microscopy, prepare and observe bacterial slides and perform staining methods and explain the mechanism of the bacteria. In order to observe and investigate microbes we need to use microscope and bacterial Staining Techniques. Microscope is the invaluable tool allows the viewing of objects or structures that otherwise would go unnoticed by human naked eyes. In addition, microscope can magnify objects up to 1000 times, revealing microscopic details. It has special techniques and optics thus it can reveal the structure and biochemistry of living cells. Microscope consists of a combination of several optical lenses. In this experiment, we are using light microscope.

Light is conducted through curve lenses in such a way that an object may be viewed larger than its actual size. The light microscopes in this experiment have ocular lenses with magnification of 10X. Moreover, there are also four different objective lenses to choose from 10X, 40X, and 100X. For Bacterial Staining Method, there are two basics techniques. One of them is using wet mount method but bacteria are too small and too transparent to be well described using light microscopy and a wet amount. Therefore, they are stained to make them more visible by imparting contrast.

Simple stain, with only one layer of cell, is coloured with different colour and methylene blue dye are used for differentiating Bacillus sp. Negative stain is particularly useful for determining cell size and arrangement and it can be used to stain cells that are too delicate to be heat-fixed. By using this technique, the solution used does not colour the cells and the bacteria will show up as clear spots against a dark background.

The gram staining Method is used as a tool for differentiation of Gram-positive and Gram-negative bacteria, as a first step to determine the identity of a particular bacterial sample. Gram-positive and Gram-negative organisms are distinguished from each other by differences in their cell walls, including the way the cell takes up and retains stains. The E. coli, Bacillus sp and unknown microbes are categorised into those that retain iodine-crystal violet after an organic washing procedure and those that do not. Gram Staining is the most consistent when done on bacteria that less than 24 hours, while the older cultures may not retain the primary stain and give inaccurate results. 2.0 Literature Review

The Essay on Osmosis in onion cell

INRODUCTION A living plant cell will shrinks or swells depending on the solute concentration of the cell in relation to the solute concentration of the fluid surrounding the cell (1). It follows that water will move from a region of high water concentration to a region of low water concentration therefore, if a cell is placed in a hypertonic solution water will move from the cell into the solution .

Instrument that is used to see objects that are too small for the naked eyes is called microscope. It can magnify objects up to 1,000 times, revealing microscopic details. With special techniques and optics the structures and biochemistry of living cells can be revealed. There is various type of microscope, the most common and first to be invented is the optical microscope which uses light to image the sample. Although, Zaccharias Janssen discovered that object appeared greatly enlarged after experimenting with several lenses in a tube in 1590 and in 1609, Galileo worked out the principles of lenses, but Anton van Leeuwenhoek is a microscope designer who first to detect microorganisms using microscope. Light microscope employs visible light to detect small objects. The biggest challenge when it comes to looking at living things are obtaining sufficient contrast, finding the focal plane, obtaining good resolution and recognizing the subjects when one sees it.

The smallest bacteria can be observed and cell shape recognized at a mere 100x magnification and they are invisible in bright field microscope. Ocular lens is a cylinder containing two or more lenses. The function is to bring the image into focus for the eye. Staining is a process in which microbes are stained to enhance contrast in the microscopic image. Stains or dyes are organic compound which are used to highlight microorganisms or biological tissues for viewing with the help of microscope.

Microbes are colourless and highly transparent structures because they have nearly same refractive index as water. Therefore, microbes cannot be seen with our naked eyes thus different types of staining methods are used to increased visibility and contrast, accentuate specific morphological features, to detect extracellular and intracellular components of microbes and preserve them for future use. The basic requirements for staining are clean grease-free slide, bacteria tobe stained, inoculating loops and Bunsen burner to sterilise inoculating loops before and after smear preparation. Two ways to fixing the slides are heat fixation and chemical fixation. Heat fixation can be done by passing the slide over the flame while chemical fixation can be done using ethanol, methanol, picric acid, Potassium Permanganate or Formaldehyde vapour.

The Term Paper on Differential Gram’s staining

. Gram stain is the most widely used staining procedure in bacteriology. It is called a differential stain since it differentiates between Gram-positive and Gram-negative bacteria. Bacteria . distilled water) Clean grease free glass slide Nichrome wire loop Dropper Filter papers Compound microscope Cedar wood oil Miscellaneous PROCEDURE: On a .

The function of fixation is to kills bacteria rendering safe handling and prevents autolysis by inactivating the autolytic enzymes. In addition, it increases the permeability of cells to stain, makes cell rigid and unfolds the globular proteins and exposing reactive groups and increasing affinity for stain. Different stains have different affinities for different organisms and they are used to differentiate different types of organisms. Bacteria are slightly negatively charged at pH 7.0 and basic dye stains bacteria while acidic dye stains background. For simple staining, only one dye is used. Simple staining is easier to perform but it has limitations. It was an easy method because only single staining agent used and either using basic or acid dyes. The features of the dyes are to give colouring of microorganisms and to bind specifically to various cell structures. For simple staining basic dyes which are positively charged are used. These dyes will attach to negatively charged

cytoplasm of microbial organism.

Negative staining is particularly useful for determining cell size and arrangement. In addition, it can be used to stains cells that are too delicate to be heat-fixed. Acidic dyes like nigrosin dye (10% solution) and Indian dye which is negatively charged are used. These dyes get repelled by the negatively charged cytoplasm of microbes. Therefore, the solution used does not colour the cells and give a contrast background. It is commonly used for determining bacteria with capsules.

Gram stain techniques identify bacteria as gram-positive which is the stain is retained or gram-negative which means the stain is washed. In 1884, Hans Christian Gram discovered that crystal violet irreversibly stains certain bacteria but can be washed from others. Gram staining can be described as a staining technique used to classify bacteria which is bacteria that are stained with crystal violet followed by a brief treatment with Gram’s iodine and after being decolourised with alcohol and treated with safranin then washed with water.

The Essay on Bacteria 2 Bacterial Cell

Describe the structure and life processes of bacteria. Bacterial cells, like plant cells, are surrounded by a cell wall. However, bacterial cell walls are made up of polysaccharide chains linked to amino acids, while plant cell walls are made up of cellulose, which contains no amino acids. Many bacteria secrete a slimy capsule around the outside of the cell wall. The capsule provides additional .

Those that retain the crystal violet are Gram-positive and those that do not retain it are Gram-negative. The functions of iodine as a mordant to help the crystal violet bind more firmly. Gram positive bacteria have the multiple layers of peptidoglycan retain the crystal violet while it is quickly rinsed out of Gram-negative bacteria because their peptidoglycan is a single layer thick. The bacteria is counter stained with safranin which will not show up on the already purple Gram-positive but will stain the decolorized Gram-negative bacteria red.

Figure 3: Cell wall difference

1) Microscope is turned on, and then the light source is adjusted. 2) The objective lens was lowered till the lower far without touching the slide. 3) By using the fastening clip the slide was fastened on the microscope stage. 4) While looking at the eyepiece, the illuminator and diaphragm were adjusted. 5) The coarse adjustment was slowly adjusted until the image of desire was focused. 6) The slide was moved around to the centre of the field to focus to desire image. 7) The slide is observed through the low power objective (x10), high dry objective (x40) and immersion oil objective (x100) for clearer view of culture. 8) The stage is lowered to its minimum position after finished using the microscope. The switch is turned off and the microscope is covered back. B) Bacterial staining techniques

I) Simple staining

1) Alcohol soaked slide are run on the flame of Bunsen burner. One drop of water was dropped to the clean slide by using sterile loop. 2) The cultures of Bacillus sp. are spread onto the water slide surface, again by using sterile loop. The loops were sterile again to kill excess microbe. 3) The slide contained by culture was passed quickly through the flame of Bunsen burner for two second for each two or three time. 4) The slide was flooded with methylene blue stain for a minute. 5) The slide is rinsed with water and blotted to dry using bibulous paper. 6) The prepared slide was examined under microscope by low power objective lens (x10), high dry power objective (x40) and oil immersion power objective (x100) to get the best view of the microorganisms. 7) The morphology of the microorganisms was drawn.

The Essay on Applying Staining Techniques To View And Identify Bacteria

. was placed on the slide so that the oil objective lens on the light microscope was employed. The bacterium was viewed and . to destroy the bacteria. From the gram stain, it was possible to determine which bacteria was gram positive or gram negative. This is . to destroy the bacteria. From the gram stain, it was possible to determine which bacteria was gram positive or gram negative. This is .

8) The procedures were repeated with bacteria E. coli.

1) Alcohol soaked slide were run on the flame of Bunsen burner. The Bacillus sp. cultures are spread onto the slide surface, by using sterile loop. The slide was set to air dry without exposed to heat. 2) Two to three of Nitrogen dye solutions were dropped to the smear. 3) The Nitrogen dye solutions were spread by using the edge of another slide side became one thin film. 4) The prepared slide was let for air dry.

5) The prepared slide was examined under microscope by low power objective lens (x10), high dry power objective (x40) and oil immersion power objective (x100) to get the best view of the microorganisms. 6) The morphology of the microorganisms was drawn.

7) The procedures were repeated with bacteria E. coli.

1) Alcohol soaked slide were run on the flame of Bunsen burner. The Bacillus sp. cultures were spread onto the slide surface, by using sterile loop. The slide was set to air dry and exposed to heat for few seconds. 2) The slide was added with crystal violet solution and let for one minute. 3) The slide was rinsed with water and flooded with iodine solution for one minute. 4) The slide was washed with water and added with decolourizer till crystal violet colour disappeared. The slide the rinsed with water. 5) Safranin was added and waits for a minute. Then the prepared slide was washed with water for maximum 5 seconds. 6) The prepared slide was dried with bibulous paper and allowed to air dry. 7) The prepared slide was examined under microscope by low power objective lens (x10), high dry power objective (x40) and oil immersion power objective (x100) to get the best view of the microorganisms. 8) The morphology of the microorganisms was drawn.

The Term Paper on Acid Fast Staining

. resist staining by ordinary methods such as a Gram stain.[1] It can also be used to stain a few other bacteria, such . methods because the slides produced by these methods can be visualized using a standard bright-field microscope. The fluorochrome method . Examine the slide with a fluorescence microscope equipped with a BG-12 exciter filter and an OG-1barrier filter. Acid-fast bacteria appear as .

9) The procedures were repeated with bacteria E. coli. and sample E2.

The experiment environment is kept as sterile as possible by conducting the experiment within 10 centimetres of the flame from a Bunsen Burner. This is to avoid contamination of the sample by microbes in the air. Gloves are worn for the same purpose. The loops used to smear the microbes onto the slide are sterilised three times in a flame. Meanwhile, the slid with sample on was to fix the bacteria onto the slide. Now about the stains, methylene blue dye is used to make the cells and nuclei more visible6. Crystal violet is used to stain the cell walls of bacteria6, which consist of peptidoglycan.

Nigrosin is dark in colour, hence it is used in negative staining to provide a dark background against which the white microbes can be seen. In Gram staining, cells are stained with crystal violet dye. Next, a Gram’s iodine solution (iodine and potassium iodide) is added to form a complex between the crystal violet and iodine. This complex is a larger molecule than the original crystal violet stain and iodine and is insoluble in water. Alcohol is added to the sample as a decolouriser, which dehydrates the peptidoglycan layer, shrinking and tightening it. The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria.

Conversely, the outer membrane of Gram negative bacteria is degraded and the thinner peptidoglycan layer of Gram negative cells is unable to retain the crystal violet-iodine complex and the colour is lost. A counterstain, safranin, is added to the sample, staining it red. Since the safranin is lighter than crystal violet, it does not disrupt the purple coloration in Gram positive cells. However, the decolorized Gram negative cells are stained red. Gram positive bacteria (with a thicker peptidoglycan layer) retain crystal violet stain during the decolourisation process, while Gram negative bacteria lose the crystal violet stain and are instead stained by the safranin in the final staining process.6 From our observation from simple staining of Bacillus sp ,it can be seen that the bacteria is rod-shaped.

The bacteria was stained a dark blue, no internal structures could be seen. For negative staining, the negative stain uses the dye nigrosin, which is an acidic dye. By giving up a proton (as an acid) the chromophore of the dye becomes negatively charged. Because the cell wall is also negatively charged only the background around the cells will become stained, leaving the cells unstained. Hence, the cells are seen as white spots against a dark background E.coli is a rod shape as well. After gram staining for E.coli, E.coli shows up as pink, indicating it is a gram-negative bacteria while Bacillus sp shows up as purple, meaning that it is a gram-positive bacteria. The unknown microbes from petri dish

1turn out to be a mixture of gram negative and gram positive bacteria because of the presence of light pink and purple regions. They are round in shape.

5.0 Questions

1) Morphology includes size, cell structure, presence of endospore and flagella. From the report, in Figure 8, the unknown microbes from petri dish 1 give purple and pink colouration after gram staining, they’re round in shape. A more reliable method to identify cell morphology would be to use special stains to identify specific parts of a microbe like endospores, which is usually present in gram positive bacteria. The method use to stain endospores is called the Schaeffer-Fulton1 method, where Malachite Green is used to stain the endospores while Safranin is a counterstain. The end result would be pink bacteria with green dots within them. 2) Three methods to characterise a microorganism include:

I) Starch hydrolysis test

This bio-chemical test is used on gram-positive bacteria to identify bacteria that can hydrolyze starch (amylose and amylopectin) using the enzymes a-amylase and oligo-1,6-glucosidase. Often used to differentiate species from the general Clostridium and Bacillus. Because of the large size of amylose and amylopectin molecules, these organisms cannot pass through the bacterial cell wall. In order to use these starches as a carbon source, bacteria must secrete a-amylase and oligo-1,6-glucosidase into the extracellular space.

These enzymes break the starch molecules into smaller glucose subunits which can then enter directly into the glycolytic pathway. In order to interpret the results of the starch hydrolysis test, iodine must be added to the agar. The iodine reacts with the starch to form a dark brown colour. Thus, hydrolysis of the starch will create a clear zone around the bacterial growth. E.g. Bacillus subtilis is positive for starch hydrolysis.2 ii) Protein analysis (gel electrophoresis, SDS-PAGE, establishment of clonality) The size and other differences between proteins among different organisms can be determined by using protein separation methods, collectively known as gel electrophoresis.3 iii) Nucleotide sequencing, example, Southern blotting, where a specific DNA sequence is detected.4 3)

Full standard procedure for operating a microscope5:

i. When moving a microscope, always carry it with both hands. Grasp the arm with one hand and place the other hand under the base for support. ii. Turn the revolving nosepiece so that the lowest power objective lens is “clicked” into position. iii. The microscope slide should be prepared with a coverslip or cover glass over the specimen. This will help protect the objective lenses if they touch the slide.

Place the microscope slide on the stage and fasten it with the stage clips. iv. Look at the objective lens and the stage from the side and turn the coarse focus knob so that the objective lens moves downward (or the stage, if it moves, goes upward).

Move it as far as it will go without touching the slide. v. Look through the eyepiece and adjust the illuminator (or mirror) and diaphragm for the greatest amount of light. vi. Slowly turn the coarse adjustment so that the objective lens goes up (away from the slide).

Continue until the image comes into focus. Use the fine adjustment, if available, for fine focusing. If the microscope has a moving stage, then turn the coarse knob so the stage moves downward or away from the objective lens. vii. Move the microscope slide around so that the image is in the center of the field of view and readjust the mirror, illuminator or diaphragm for the clearest image. viii. Then change to the next objective lens with only minimal use of the focusing adjustment. Use the fine adjustment, if available. If you cannot focus on your specimen, repeat steps 4 through 7 with the higher power objective lens in place. ix.

The proper way to use a monocular microscope is to look through the eyepiece with one eye and keep the other eye open (this helps avoid eye strain).

x. Do not touch the glass part of the lenses with your fingers. Use only special lens paper to clean the lenses. xi. When finished, lower the stage, click the low power lens into position and remove the slide. xii. Always keep your microscope covered when not in use. Dust is bad for the microscope. 4) Three accidents that can occur during the experiment are: i. A sleeve catching fire while passing the slide through the flame in wide sweeping motions ii. the slide dropping due to a weak grip on it

The slide breaking due to over-exposure to fire and a strong grip.

The staining techniques in this experiment are the correct way to identify the shape and size of the bacteria. Bacillus sp is rod-shaped and gram positive while E.coli is rod-shaped and gram negative. The unknown microbes from petri dish 1 are a mixture of gram-positive and gram-negative bacteria and are round.

7.0 Recommendations

It is suggested that a smaller amount of microbes are smeared on the glass slide to prevent the sample from looking so dense under the microscope, thus preventing us from seeing the shape and size clearly. Next, increase the amount of the negative stain to ensure more visibility of the cells under the microscope. Lastly, clean the lens of the microscope before use to avoid confusing images in the eyepiece.

References

1) Endospores, retrieved from: http://pscantie.myweb.uga.edu/stain.html 2) Rachel Watson M.S., retrieved from: http://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm#starch 3) Stephen T. Abedon, 6th July 1999, retrieved from: http://www.mansfield.ohio-state.edu/

sabedon/biol3010.htm 4) McGraw-Hill, retrieved from: http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/bio_g.swf::Southern%20Blot 5) How to Use a Microscope, retrieved from: http://www.microscope-microscope.org/basic/how-to-use-a-microscope.htm 6) Monica Z. Bruckner,Microbial Life Edcucational resources , retrieved from : http://pscantie.myweb.uga.edu/stain.html 7) Bio-imaging, 2004, retrieved from: http://web.path.ox.ac.uk/

bioimaging/bitm/instructions_and_information/EM/neg_stain.pdf 8) Staining, retrieved from: en.wikipedia.org/wiki/staining

9) Definition of staining, retrieved from: www.thefreedictionary.com/staining 10) Monica Z. Bruckner, Gram Staining, retrieved from: serc.caleton.edu/microbiolife/research_methods/microscopy/gramstain.html 11)

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