MLT 415 - Lab Report (Gram stain techniques) PDF

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MLT 415 – Fundamentals of Microbiology Objectives: 1. To know the principle of Grain stain. 2. To practice Gram stain on the given smear. 3. To report the result of the unknown smear. Principles: Staining is an auxiliary technique used in microscopic techniques used to enhance the clarity of the mic...

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MLT 415 – Fundamentals of Microbiology

Objectives: 1. To know the principle of Grain stain. 2. To practice Gram stain on the given smear. 3. To report the result of the unknown smear.

Principles: Staining is an auxiliary technique used in microscopic techniques used to enhance the clarity of the microscopic image. Gram-staining was firstly introduced by Cristian Gram in 1883 which used to differentiate the Gram-positive microorganisms and Gram-negative microorganisms. The procedure is based on the ability of microorganisms to retain colour of the stains used during the gram stain reaction. Gram-positive microorganisms stain blue-purple and Gram-negative microorganisms stain pink-red. This is because Gram-negative microorganisms are decolorized by the alcohol which causes it losing the colour of the primary stain dye, purple. Grampositive microorganisms are not decolorized by alcohol which means the colour will remain as purple. After decolourization step, a colourless gramnegative microorganism’s will able to accept counterstain colour which usually in pink colour. The

Gram

stain

is

a

very

important

step

in

the

initial

characterization and classification of microorganisms. This is because the different type of microorganisms have different characteristic especially the thickness of the cell wall. The peptidoglycan layer in Gram-positive microorganisms

is

thicker

than

in

Gram-negative

microorganisms.

Peptidoglycan is mainly a polysaccharide composed of two sub-units called N-acetyl glucosamine and N-acetyl muramic acid. The thick peptidoglycan layer of Gram-positive organisms allows these organisms to retain the crystal violet-iodine complex and stains the cells as purple. The microorganisms present in an unstained smear are invisible when viewed Page | 1

MLT 415 – Fundamentals of Microbiology

using a light microscope. Therefore, the microorganisms need to be in stained smear so the morphology and arrangement of the bacteria can be observed as well. There are two type of step in Gram stain techniques which are three-step Gram stain and four-step Gram stain. However, most clinical laboratories are currently using the four step Gram stain. The colour of the microorganisms that can be seen under the light microscope can be either purple or red-pink with the background of the tissue is yellow. The most common arrangement in the stained smear is either in chain or in cluster. However it is only be used to describe the Gram-positive microorganisms and there is no significant to used it to describe the Gram-negative microorganisms. There are three types of common morphologies that usually found in the stained smear. 1) Cocci  May occur in pairs or in group of four, in grape like cluster, in

chain or in cubical arrangement. 2) Bacilli  Rod-shaped bacteria which generally occur singly but not

occasional be found in pairs or chains. 3) Cocco-bacilli  Elongated spherical or ovoid form bacteria.

GramStain

None

Crystal-

Gram’s

Acetone

Violet

Iodine

Alcohol

Safranin

Reagent

GramPositive Organisms

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MLT 415 – Fundamentals of Microbiology

GramNegative Organisms

Table 1: Illustration on the colour changes of microorganisms in the staining process

Materials/Equipment: 1. 2. 3. 4. 5.

Two Unstained Smear; Staphylococcus aureus and Escherichia coli Light Microscope Immersion Oil Electrical Incinerator Gram stain reagent i. Primary stain (Crystal Violet) ii. Mordant (Gram’s Iodine) iii. Decolourizer (Acetone Alcohol) iv. Counterstain (Safranin)

Procedures 1. Smears from cultures of at two type microorganisms were prepared. 2. The slides on a staining rack and flood each smear were placed with 3. 4. 5. 6.

crystal violet for 1 minute. The crystal violet from each slide was wash with tap water. Each smear was flooded with Gram’s iodine for 1 minute. The Gram’s iodine from each slide was washed with tap water. Each slide was decolorized with acetone alcohol until the slide

appears colourless for 5 to 15 seconds. 7. The slide was briefly washed with tap water. 8. The smear was counterstained with safranin for about 1 minute. 9. The smear was briefly washed with tap water and blot dry. 10. The slides were examined under the microscope.

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MLT 415 – Fundamentals of Microbiology

Results 

Smear 1: S. aureus

Figure 1: Gram-positive bacteria

Observation: Gram positive cocci in cluster.



Smear 2: E. coli

Figure 2: Gram-negative bacteria

Observation: Gram negative bacilli.

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MLT 415 – Fundamentals of Microbiology

Discussion In this experiment, we focused on gram staining mechanisms. Firstly,

we

prepared

smears

of

two

microorganisms

which

are

Staphylococcus aureus and Escherichia coli. Theoretically, both of these microorganisms are from two different groups, therefore we expected to observe two different results in this gram staining experiment. We stain both smears with crystal violet dyes. Crystal violet dyes have positively charged particles that penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. Then the positively particles bind to negatively charged molecule at the cell wall of bacteria and stains the bacterial cells purple. 1 minute later, the crystal violet was washed with tap water and then the slides are dried. The next step is to add Gram’s iodine onto each smear. Iodine was acted as a mordent and trapping agent to form crystal violet-iodine complex. A mordant is a substance that increases the affinity of the cell wall, thus forming an insoluble complex which gets trapped in the cell wall. This complex enables the dyes to not be easily being removed. This is because in the Gram stain reaction, the crystal violet and iodine form an insoluble complex which serves to turn the smear a dark purple colour. At this stage, all cells will turn purple. Next, we washed the Gram’s iodine with tap water. Acetone alcohol was used as decolorizing agent that dissolves the lipid outer membrane of Gram negative bacteria, thus leaving the peptidoglycan layer exposed and increases the porosity of the cell wall. The crystal violet-iodine complex then washed away from the thin peptidoglycan layer, leaving Gram negative bacteria colourless. For Grampositive bacteria, the addition of alcohol dehydrated the layer of peptidoglycan which in turn would trap the crystal violet-iodine complex. This cause the Gram-positive bacteria appeared to be in purple colour. The decolourization step must be performed carefully, otherwise overdecolourization may occur. If the decolourizing agent is applied on the cell Page | 5

MLT 415 – Fundamentals of Microbiology

for too long time, the Gram-positive organisms will appear as Gramnegative organisms and if the decolourizing agent is not applied on the cell long enough, resulting in Gram-negative bacteria to appear as Grampositive bacteria.

The decolorized Gram negative cells can be rendered visible with a suitable counterstain. Safranin was used to counterstain both smears. This is to enables the Gram-negative bacteria to be visualized easily as it can be stain in pink colour. The Gram-positive bacteria does not being stained pink when safranin was being introduced because the peptidoglycan layer already have crystal violet-iodine complex. Finally, we observed our specimen using microscope under oilimmersion objective lenses. This particular lens has more mirrors inside and it requires the use of oil to refract light rays towards the centre of the lenses. The result shows that the smear named S. aureus is a Gram-positive organism because it shows a purple in colour while the E. coli is Gramnegative organism because it shows a pink-red in colour.

Conclusion From the experiment, the gram staining is the method to distinguish and differentiate between Gram-positive bacteria and Gram-negative bacteria. In this experiment, the use of proper materials to help us for reaching the aim of this experiment such as Crystal violet, Gram’s iodine, acetone alcohol, safranin, and microscope slide. In the end of experiment it shows that S. aureus and E. coli has different thickness of cell wall, Page | 6

MLT 415 – Fundamentals of Microbiology

different morphology and arrangement.. The S. aureus has morphology of a cocci with a cluster arrangement as shown in Figure 1 while the morphology of E. coli is Gram-negative organisms is bacilli as shown in Figure 2. In order to achieve satisfied result, there are several procedures that we have to do in order to avoid the error in this experiment, such as prepare smear from cultures of microorganism, place the slides on a staining rack, and so on.

References 1. Prescott, L. (2002). Prokaryotic Cell Structure and Function. In Microbiology (5th ed.,

pp.

60-61).

New

York,

NY

10020:

The

McGraw-Hill companies. 2. Willey, J. (2009). The Diversity of the Microbial World. In Prescott's Principles of

Microbiology (pp. 474-499). New York, NY 10020:

The McGraw-Hill companies 3. Goering, R. (2008). Microbes as parasites. In Mim's Medical Microbiology (4th ed., pp. 10-11). UK: Mosby-Year Book Europe. 4. Brown E. Alfred, Benson’s Microbiological Applications, Ninth edition, McGraw Hill Publication. 5. Cappucino G. James, Sherman Natalie, Microbiology A laboratory manual, Seventh edition, Pearson Education. 6. Akbar Haqi, Microbiology 3 Laboratory Gram Staining, (2007), Academia.edu.

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