IBG 102 LAB REPORT

Before we continue our lab reports, we would like to introduce ourselves first.

Here we go, here are our group members

 Hi, I'm Tiam Meng^^ !!!
















                                                             
                                                                                                                                              

Hi, I'm Iskandar.

 Hi, I'm Azizul

Hi, I'm Aizat
















Okay, let's begin with our lab reports.

Lab 1 Report Written by Yu Tiam Meng

Name : Yu Tiam Meng

Matrix number : 111437

LAB 1 : PRINCIPLES AND USE OF MICROSCOPE

1.1 Setting up and using the microscope

Introduction:
In order to be seen, microorganisms need to be magnified. Despite advances in other area of microscopy (for example, the electron microscopy), the light microscopy is still the instrument most frequently used for viewing microorganisms.

Objective:
Learn to use a simple bright-field microscope correctly.

Results:
Total magnification = objective lens power x eyepiece lens power(10x)

Diplococcus under 40x magnification:

Diplococcus under 100x magnification:

Diplococcus under 400x magnification:

Discussions:
1) The light intensity is adjusted to make sure that the pictures captured are clear and not too bright.

2) Colony morphology of Diplococcus: A diplococcus is a round bacterium that typically occurs in the form of two joined cells. Its name comes from diplo, meaning double, and coccus, meaning berry.

Conclusion:

I managed to get the view of specimen by adjusting the fine and coarse adjustment knob but I forgot to copy down the full name of specimen.

References:

http://en.wikipedia.org/wiki/Bacterial_cellular_morphologies

1.2 Examination of cells

Introduction :
Because of their extreme minuteness, bacteria are not generally studied with the low-power or high-power dry objectives. Instead they are stained and observed with the oil immersion objective.

The wet mount methods enables you to study the sizes and shapesof living microorganisms (drying or staining microorganism distort them). It also enables you to determine if cells are motile. The wet mount method is quick and easy, and does not require special equipment.

Objective     :
-To provide an experience in the use of microscope.

-To illustrate the diversity of cells and microorganisms.

Result          :

Under 100x objective X 10x eyepiece = 1000x magnification

1.Saccharomyces cerevisiae (Yeast)

2.Lactobacillus fermentum

Discussions:

1) Oil immersion is a technique used to increase the resolution of a microscope. This is achieved by immersing both the objective lens and the specimen in a transparent oil of high refractive index, thereby increasing the numerical aperture of the objective lens.

2)Colony morphology of Lactobacillus fermentum: 
 - They ferment sugars and produce lactic acid and they are aerotolerant anaerobes(they grow fermentatively with or without oxygen)
 - They are in rod shape, tiny in size, smooth surface, and violet in colour.

3)Colony morphology of Saccharomyces cerivisiae:
  - Yeast are nonfilamentous, unicellular fungi that are typically spherical or oval.
  - Yeast are capable of facultative anearobic growth. 
  - Mycelium absent. Vegetative cells reproducing by multilateral budding, without a mucous coating. Asci morphologically similar to vegetative cells, not in well defined-chains, thin-walled, 1- to 4-spored, evanescent or semi-persistent, without an obvious discharge mechanism. Ascospores usually spherical, oftten ornamented with equatorial ridges. Fermentation positive, coenzyme usually Q-6.

 
 
  

Conclusion:
The use of immersion oil and 1000x magnification enable me to get a clearer image of specimens.

References:

1) http://en.wikipedia.org/wiki/Oil_immersion

2) MICROBIOLOGY (AN INTRODUCTION) FOURTH EDITION by Tortora , Funke , Case , 1992 by The Benjamin/Cummings Publishing Company, Inc.
3) Introduction to Microbiology (Second Edition) by John L. Ingraham/Catherine A. Ingraham, 2000 by Brooks/Cole.

LAB 1 REPORT WRITTEN BY ISKANDAR

Introduction:

- microorganisms need to be seen in a microscope. 1.1 Introduction to Microscope

Microscope is an instrument that is used to observed sample of microorganisms under high magnification. Light microscope is type of microscope that is frequently used for viewing microorganisms. A brightfield microscope is a type of microscope that utilizes the brightfield microscopy technique.  This is a type of optical microscopy illumination that is the simplest and most reliable among all the microscopy techniques out there.  This microscopy technique uses white light for illumination that comes from below the specimen in what is called transmitted illumination.However, it can also be considered as the method of lighting in a stereoscope using upper incident illumination.

Utilizing the most basic type of microscopy, the bright field microscope is widely used from simple and starting types like kid or child school microscope to university or professional level models. Just as a simple compound light microscope that uses brightfield microscopy is important to students as well as children, bright field microscopes are also indispensable to biologists as well as microbiologists in their respective fields.

There is 4 different magnification of objective lenses. That is 4x magnification, 10x magnification, 40x magnification and 100x magnification. The objective lens focuses the light passing through the specimen to form a magnified primary image. There is also an eyepiece tube that receives light coming through the objective lens and redirects it to the eyepiece. Magnification of the eyepiece is 10x magnification. The eyepiece consists of several lenses that collect the light, focus it and transmit it to the eye. Total image of magnification is calculated by multiplying the objective lens power multiplication by the eyepiece lens power multiplication.

The resolving power of resolution of microscope is its ability to distinguish two very small and closely spaced objects as separate entities. A number of factors affect resolution is the condenser diaphragm. Closing the diaphragm image contrast but decreased the resolution, opening the diaphragm decreases the contrast but increased the resolution.

Eyepiece or Ocular is what you look through at the top of the microscope. Typically, standard eyepieces have a magnifying power of 10x. Optional eyepieces of varying powers are available, typically from 5x-30x.

Eyepiece tube holds the eyepieces in place above the objective lens. Binocular microscope heads typically incorporate a diopter adjustment ring that allows for the possible inconsistencies of our eyesight in one or both eyes. The monocular (single eye usage) microscope does not need a diopter. Binocular microscopes also swivel (Interpupillary Adjustment) to allow for different distances between the eyes of different individuals.

Objective Lenses are the primary optical lenses on a microscope. They range from 4x-100x and typically, include, three, four or five on lens on most microscopes. Objectives can be forward or rear-facing.

Nosepiece houses the objectives. The objectives are exposed and are mounted on a rotating turret so that different objectives can be conveniently selected. Standard objectives include 4x, 10x, 40x and 100x although different power objectives are available.

Coarse and Fine Focus knobs are used to focus the microscope. Increasingly, they are coaxial knobs - that is to say they are built on the same axis with the fine focus knob on the outside. Coaxial focus knobs are more convenient since the viewer does not have to grope for a different knob.

Stage is where the specimen to be viewed is placed. A mechanical stage is used when working at higher magnifications where delicate movements of the specimen slide are required.

Stage Clips are used when there is no mechanical stage. The viewer is required to move the slide manually to view different sections of the specimen.

Aperture is the hole in the stage through which the base (transmitted) light reaches the stage.

Illuminator is the light source for a microscope, typically located in the base of the microscope. Most light microscopes use low voltage, halogen bulbs with continuous variable lighting control located within the base.

Condenser is used to collect and focus the light from the illuminator on to the specimen. It is located under the stage often in conjunction with an iris diaphragm.

Iris Diaphragm controls the amount of light reaching the specimen. It is located above the condenser and below the stage. Most high quality microscopes include an Abbe condenser with an iris diaphragm. Combined, they control both the focus and quantity of light applied to the specimen.

Condenser Focus Knob moves the condenser up or down to control the lighting focus on the specimen.

Objective:-

To learn a simple bright field microscope correctly.

Material and reagents:

Microscope slide and cover slip

Procedure:

Setting up:

Sit comfortably on stool and knee under the bench and move the microscope to look to both eyepieces without any strain.

Microscope is turned on by using main switches.

By using the brightness control, the light intensity is adjusted until position 5.

Nosepiece is rotated until 4x objective lens is brought to the light path.

Clean slide with a mark on it is placed on the stage and is secured with a spring clip. By using coaxial stage control knobs, the slides is moved to the light path.

Both eyepieces is adjusted until a single circle of light can be seen.

Tube length adjustment (diopter) ring on right eyepiece was rotated until it match the interpupillary.

By using right eye only, slide with the mark is adjusted with coarse and fine adjustment knobs until it is focused.

By using left eye only, slide with the mark is adjusted with coarse and fine adjustment knobs until it is focused.

Low power (10x) objective viewing:

Slide that is marked with marker pen is replaced with specimen slide.

Stage is moved to obtained view of specimen and focus is adjusted by using fine adjustment knob. Then 10x objective lens is changed.

By placing an object in the centre of the glass above light source, the condenser is focused by adjusting the light condenser.

Object is removed out of focused by lowering the condenser sufficiently.

Eyepiece is replaced and re-focused with fine adjustment to obtained better image from specimen with poor contrast.

High power (40x) objective viewing :

Specimen was focused with 10x objective lens. From side of microscope, 40x objective lens is changed.

Condenser is raised within 1cm of slide.

Specimen is focused by using fine adjustment knob, light intensity is adjusted using brightness control if necessary.

Condenser diaphragm is adjusted for optimum contrast.

Oil immersion (100x) objective viewing :

Specimen was focused with 40x objective lens. From side of microscope, 100x objective lens is changed and objective lens is not allow to touch the slide.

Objective lens is carefully turned to one side and drops of oil is placed onto the slide. Objective lens is turned again until it is in the light path.

Condenser is raised as closed to the slide as possible.

Specimen is focused by using fine adjustment knob, light intensity is adjusted using brightness control if necessary.

Condenser diaphragm is adjusted for optimum contrast.

After used :

Specimen slide is removed and discarded into appropriate discard container.

Light brightness control is reset to its lowest setting.

Lowest power objective is reset to working position.

Oil from the 100x objective lens is cleaned by using lens tissue.

Microscope is turned off from the switch and power point, cord is disconnect and carefully wrapped around base of microscope.

Cover is replaced.

Care of microscope :

Microscope is carried by holding it firmly at the arm and support from the base. The instrument is kept upright.

Never placed the microscope at the edge of bench.

Only specified lens tissue is used to clean the lenses.

Cover slip is used when examining object or organism that is mounted with water or other fluids.

Stage was always lowered when placing or removing slide.

Lowest power objective lens was always placed in working position after the microscope had been used.

What do we observe is=

The following picture is 1) sample with 4x10 magnification. 2) sample with 10x10 magnification 3) sample with 40x10 magnification.

1.2 examination of cells

Introduction

Because of their extreme minuteness ,bacteria are not generally studied with the low power or high power dry objects. Instead they are stained and observed with the oil immersion objective.

The wet mount methods enables you to study the size and shapes of living microorganism. It also enables you to determine if the cell are motile. The wet mount method is quick and easy and does not require any special equipment.

Objective

-          To provide an experience in the use of microscope.

-          To illustrate the diversity of cells and microorganism.

Material and reagents

-          Culture

-          Immersion oil

-          Lens tissue

-          A microscope slide containing stained microorganism

-          Inoculating loop

-          Bunsen burner

-          Slip and cover slip.

Procedure

Stained cells:

-          The microscope is setted up as described and the slide is examined under the oil immersion lens

-          The shape and size of the organism is observed. Picture are been captured.

The wet mount:

-          A sterile pasteur pipette is used to aseptically transfer one drop of culture to the centre of the glass slide.

-          A marker pen is used to mark the cover slip.

-          The cover slip is taken and turned upside down so that the marker pen is faced down.

-          The slide on the microscope stage is placed and 4x objective focus is used on the culture.

-          The cell is observed using 10x and 40x objectives.

-          The cells is observed by using oil immersion.

-          Pictures has been captured.

-          The process is repeated with other cultures.

Observation:

Stained cell:-

                                               Stained cells slide 4x10 magnification

                                                   Stained cell 10x10 magnification

                                                     Stained cell 40x10 magnification

Wetted mount.

        Saccharomyces cerevisea sp. Under 100x10 magnification under oil immersion.

Lactobacillus fermentum sp. Under 100x10 magnification under oil immersion.

Discussion:

Stained Cell

Bacteria are stain so that they can be more easily visualized under the microscope. Some stains can also be used to identify and classify bacteria. Gram stain is a method of staining bacteria using a dye called crystal (gentian) violet. Gram's method helps distinguish between different types of bacteria species into two large groups.

The gram-staining characteristics of bacteria are denoted as Gram-positive or Gram-negative, depending upon whether the bacteria take up and retain the crystal violet stain or not. The Gram stain is almost always the first step in the identification of a bacterial organism, and is the default stain performed by laboratories over a sample when no specific culture is referred. A Gram positive results in a purple/blue color while a Gram negative results in a pink/red color.

Gram-positive bacteria retain the color of the crystal violet stain in the Gram stain. This is characteristic of bacteria that have a cell wall composed of a thick layer of a particular substance, specificallypeptidologlycan containing teichoic and lipoteichoic acid complexed to the peptidoglycan. Molecules of crystal violet combine with iodine molecules to form a complex within the microbial cell. The addition of decolorizer effectively dehydrates the peptidoglycan layer preventing the extraction of the crystal violet–iodine complex from the interior of the cell. The final step consists of the addition of safranin counterstain. The color from the retained crystal violet-iodine complex obscures the safranin counterstain to give the cells a dark blue, or purple, appearance via high resolution light microscopy.

The Gram-positive bacteria include staphylococci ("staph"), streptococci ("strep"), pneumococci, and the bacterium responsible for diphtheria (Cornynebacteriumdiphtheriae) and anthrax (Bacillus anthracis).

Gram-negative bacteria lose the crystal violet stain (and take the color of the red counterstain) in Gram's method of staining. This is characteristic of bacteria that have a cell wall composed of a thin layer of a particular substance (specifically, peptidoglycan covered by an outer membrane of lipoprotein and lipopolysaccharide containing endotoxin). Molecules of crystal violet combine with iodine molecules to form a complex within the microbial cell. The addition of Gram decolorizer effectively extracts the outer membrane and dehydrates the peptidoglycan layer. It is presumed that the inner membrane is extracted by the Gram decolorizer, but remains associated with the mureinsacculus . However, the thinner dehydrated peptidoglycan layer cannot prevent the extraction of the crystal violet–iodine complex from the interior of the cell through the peptidoglycan layer. The final step consists of the addition of safranin counterstain. As the crystal violet-iodine complex has been extracted from the cells, the safranin counterstain gives the cells a pink, or red, appearance via high resolution light microscopy.

As for the lactobacillus fermentum, the shape is rod shape.mostly different length and the colour are transluscent. The elevation cannot be recognize and the size are punctiform. The surface of the bacteria mentioned are smooth and shiny.

As for the saccharomyces cerevisea the shape is almost circular and we can see the nucleus and some of the nucleus are undergoing mitosis and there is no elevation and the suface of the bacteria should be shinny and smooth.

References:

1) http://en.wikipedia.org/wiki/Oil_immersion

2) MICROBIOLOGY (AN INTRODUCTION) FOURTH EDITION by Tortora , Funke , Case , 1992 by The Benjamin/Cummings Publishing Company, Inc.
3) Introduction to Microbiology (Second Edition) by John L. Ingraham/Catherine A. Ingraham, 2000 by Brooks/Cole.

LAB 1 WRITTEN BY AZIZUL

Name : Ahmad Azizul Bin Md Sadik
Matrix No : 114116

LAB 1 : PRINCIPLES & USED OF MICROSCOPE

1.1 Introduction to Microscope

Microscope is an instrument that is used to observed sample of microorganisms under high magnification. Light microscope is type of microscope that is frequently used for viewing microorganisms. A brightfield microscope is a type of microscope that utilizes the brightfield microscopy technique.  This is a type of optical microscopy illumination that is the simplest and most reliable among all the microscopy techniques out there.  This microscopy technique uses white light for illumination that comes from below the specimen in what is called transmitted illumination.However, it can also be considered as the method of lighting in a stereoscope using upper incident illumination.

Utilizing the most basic type of microscopy, the bright field microscope is widely used from simple and starting types like kid or child school microscope to university or professional level models. Just as a simple compound light microscope that uses brightfield microscopy is important to students as well as children, bright field microscopes are also indispensable to biologists as well as microbiologists in their respective fields.

There is 4 different magnification of objective lenses. That is 4x magnification, 10x magnification, 40x magnification and 100x magnification. The objective lens focuses the light passing through the specimen to form a magnified primary image. There is also an eyepiece tube that receives light coming through the objective lens and redirects it to the eyepiece. Magnification of the eyepiece is 10x magnification. The eyepiece consists of several lenses that collect the light, focus it and transmit it to the eye. Total image of magnification is calculated by multiplying the objective lens power multiplication by the eyepiece lens power multiplication.

The resolving power of resolution of microscope is its ability to distinguish two very small and closely spaced objects as separate entities. A number of factors affect resolution is the condenser diaphragm. Closing the diaphragm image contrast but decreased the resolution, opening the diaphragm decreases the contrast but increased the resolution.

Eyepiece or Ocular is what you look through at the top of the microscope. Typically, standard eyepieces have a magnifying power of 10x. Optional eyepieces of varying powers are available, typically from 5x-30x.

Eyepiece tube holds the eyepieces in place above the objective lens. Binocular microscope heads typically incorporate a diopter adjustment ring that allows for the possible inconsistencies of our eyesight in one or both eyes. The monocular (single eye usage) microscope does not need a diopter. Binocular microscopes also swivel (Interpupillary Adjustment) to allow for different distances between the eyes of different individuals.

Objective Lenses are the primary optical lenses on a microscope. They range from 4x-100x and typically, include, three, four or five on lens on most microscopes. Objectives can be forward or rear-facing.

Nosepiece houses the objectives. The objectives are exposed and are mounted on a rotating turret so that different objectives can be conveniently selected. Standard objectives include 4x, 10x, 40x and 100x although different power objectives are available.

Coarse and Fine Focus knobs are used to focus the microscope. Increasingly, they are coaxial knobs - that is to say they are built on the same axis with the fine focus knob on the outside. Coaxial focus knobs are more convenient since the viewer does not have to grope for a different knob.

Stage is where the specimen to be viewed is placed. A mechanical stage is used when working at higher magnifications where delicate movements of the specimen slide are required.

Stage Clips are used when there is no mechanical stage. The viewer is required to move the slide manually to view different sections of the specimen.

Aperture is the hole in the stage through which the base (transmitted) light reaches the stage.

Illuminator is the light source for a microscope, typically located in the base of the microscope. Most light microscopes use low voltage, halogen bulbs with continuous variable lighting control located within the base.

Condenser is used to collect and focus the light from the illuminator on to the specimen. It is located under the stage often in conjunction with an iris diaphragm.

Iris Diaphragm controls the amount of light reaching the specimen. It is located above the condenser and below the stage. Most high quality microscopes include an Abbe condenser with an iris diaphragm. Combined, they control both the focus and quantity of light applied to the specimen.

Condenser Focus Knob moves the condenser up or down to control the lighting focus on the specimen.

1.2 Examination of Cells

Introduction

Historically, the study of cell biology could not have happened without the invention of microscopes because cells were not known to exist before Antonin van Leeuwenhoek and Robert Hooke saw them in their primitive microscopes Today, much cell biology research still requires careful microscopic examination of cells and their internal structures. It is not too strong a statement to say that microscopy is the single most important tool for the cell biologist.

Because of the extreme minuteness, bacteria are not generally studied with the low-power or high-power dry objectives. Instead it was stained and observed with the oil immersion objective.

The wet mount methods enables you to study the sizes and shapes of living microorganisms. It also enables to determine whether the cells are motile. The wet mount is quick and easy, also it does not require any special equipment.

Objectives :

To provide an experience in the use of microscope.

To illustrate the diversity of cells and microorganisms.

Materials and Reagents :

Culture

Immersion oil

Lens tissue

Microscope slide containing microorganisms

Inoculating loop

Bunsen burner

Slide and coverslip

Observations :

Sample for stained cell, Staphylococcus aureus :

Observation under 4x10 magnification. 

Observation under 10x10 magnification.  

Observation under 40x10 magnification.  

Sample for wet mount :

Observation under 100x10 magnification (oil immersion) for yeast sample, Saccharomyces cerevisiae.

Observation under 100x10 magnification (oil immersion) for Lactobacillus fermentum.

Discussion :

Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet. Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with. Alternatively, Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decoloring process.

The Gram stain also used to detect the presence of bacteria, yeast, and other cells in direct smears prepared from swabs, aspirates, secretions, etc. from any part of the body where infection is suspected. Direct smears are often made of throat swabs, sputum, genital swabs, wounds, abscesses, cerebrospinal fluid (CSF), serous fluids, joint fluid, urine, and stool. Gram stain is also performed to help identify colonies isolated from cultures. In addition to gram-negative or gram-positive, organisms are evaluated for size, shape, arrangement, number, and any special characteristics such as bipolar staining and the presence of spores. These characteristics often point the way to the most efficient selection of biochemical tests needed to identify the organism. The finding of organisms on direct examination of some specimens is sufficient to establish a preliminary diagnosis and justify immediate antibiotic treatment pending confirmation by culture or other means. The Gram stain is very useful in identifying anaerobic bacteria by comparing the microscopic morphology and number of organisms to culture results. Significant numbers of characteristic bacteria on Gram stain not appearing on aerobic culture often signals the presence of an anaerobic infection.

Staphylococcus aureus forms a fairly large yellow colony on rich medium. S. aureus is often hemolytic on blood agar. Staphylococci are facultative anaerobes that grow by aerobic respiration or by fermentation that yields principally lactic acid. The bacteria are catalase-positive and oxidase-negative. S. aureus can grow at a temperature range of 15 to 45 degrees and at NaCl concentrations as high as 15 percent. S. aureus should always be considered a potential pathogen. Staphylococci are perfectly spherical cells about 1 micrometer in diameter. The staphylococci grow in clusters because the cells divide successively in three perpendicular planes with the sister cells remaining attached to one another following each successive division. Since the exact point of attachment of sister cells may not be within the divisional plane, and the cells may change position slightly while remaining attached, the result is formation of an irregular cluster of cells. 

The shape and configuration of the Gram-positive cocci helps to distinguish staphylococci from streptococci. Streptococci are slightly oblong cells that usually grow in chains because they divide in one plane only, similar to a bacillus. Without a microscope, the catalase test is important in distinguishing streptococci (catalase-negative) from staphylococci, which are vigorous catalase-producers. The test is performed by adding 3% hydrogen peroxide to a colony on an agar plate or slant. Catalase-positive cultures produce O₂ and bubble at once. The test should not be done on blood agar because blood itself contains catalase. 

Lactobacillus fermentum is a Gram-positive species of bacterium in the genus Lactobacillus. It is associated with active dental caries lesions. It is also commonly found in fermenting animal and plant material. It has been found in sourdough. A few strains are considered probiotic or "friendly" bacteria in animals and at least one strain has been applied to treat urogenital infections in women. Lactobacillus fermentum belongs to the genus Lactobacillus. Species in this genus are used for a wide variety of applications. These applications include food and feed fermentation. It has been found that some strains for Lactobacillus fermentum have natural resistances to certain antibiotics and chemotherapeutics. They are considered potential vectors of antibiotic resistance genes from the environment to humans or animals to humans. A microorganism is considered a probiotic by meeting certain characteristics, such as being of human origin, non-pathogenic, having high resistance to passing through the intestine, and being beneficial to the immune system. In general, they are seen as beneficial to the host’s body and the human health. Lactobacillus fermentum has been identified as potential probiotic. The use of gut microbes as probiotics in food is aimed towards preventing and treating various health problems. Among these health problems allergies, neoplastic growth, and inflammatory bowel disease are included. Recent areas of study have focused on the influence of probiotics on metabolic functions of their host. One area has been the metabolism of cholesterol by LABs acting as probiotics. Research has shown that Lactobacillus species have been proven to remove cholesterol in vitro through various ways such as assimilation, binding to the surface cells, and incorporation into cellular membranes.

Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes (one can see the yeast as a component of the thin white film on the skins of some dark-colored fruits such as plums; it exists among the waxes of the cuticle). It is one of the most intensively studied eukaryotic model organisms inmolecular and cell biology, much like Escherichia coli as the model bacterium. It is the microorganism behind the most common type of fermentation. S. cerevisiae cells are round to ovoid, 5–10 micrometres in diameter. It reproduces by a division process known as budding. Many proteins important in human biology were first discovered by studying their homologs in yeast; these proteins include cell cycle proteins, signaling proteins, and protein-processing enzymes. The petite mutation in S. cerevisiae is of particular interest. Saccharomyces cerevisiae is currently the only yeast cell that is known to have Berkeley bodies present, which are involved in particular secretory pathways.

Although bacterial cells are much smaller and simpler in structure than eukaryotic cells, the bacteria are an exceedingly diverse group of organisms that different in size, shape, habitat, and metabolism. Much of the knowledge about bacteria has come from studies of disease-causing bacteria, which are more readily isolated in pure culture and more easily investigated than are many of the free-living species of bacteria. It must be noted that many free-living bacteria are quite different from the bacteria that are adapted to live as animal parasites. Thus, there are no absolute rules about bacterial composition or structure, and there are many exceptions to any general statement.

Conclusion :

As the conclusion, the experiment has provided the knowledge on how to use a microscope and how to handle it. Bright field type of microscope is used as the part of the experiment in observing a sample. Readily stained sample and wet mount sample is observed under a magnification of 40x, 100x, 400x and 1000x (oil immersion) magnification. Other than the used of microscope, the diversity of cells and microorganisms can be illustrated from the observation of the sample.

References :

1.    http://www.enotes.com/gram-stain-reference/gram-stain

2.    http://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html

3.    http://textbookofbacteriology.net/staph.html

4.    http://en.wikipedia.org/wiki/Lactobacillus_fermentum

5.    http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae

6.    http://www.britannica.com/EBchecked/topic/48203/bacteria/39334/Diversity-of-structure-of-bacteria

7.    http://www.brightfieldmicroscopes.com/parts-of-a-brightfield-microscope.html

8.    http://www.microscope.com/compound-microscope-parts-t-7.html

LAB 1 WRITTEN BY AIZAT

Name : Muhammad Aizat B Mat Saad
Matrix No : 111385

LAB 1: PRINCIPLES AND USE OF MICROSCOPE

INTRODUCTION:

The magnification of small things is a necessary facet of biological research, but the fine detail in cells and in subcellular components requires that any imaging system be capable of providing spatial information across small distances. Resolution is defined as the ability to distinguish two very small and closely-spaced objects as separate entities. Resolution is best when the distance separating the two tiny objects is small. The usefulness of any microscope is that it produces better resolution than the eye. Microscope is the instrument that produces enlarged images of small objects that are invisible to our naked eye, allowing the observer an exceedingly close view of minute structures at a scale convenient for examination and analysis.The microscopes fit into two categories: light (or optical) and electronic, depending on the principle or method of magnification employed. In light microscopy, the magnification is obtained through a system of optical lenses while in electronic microscopy a bean of electrons is used to produce the enlarged image.The microscope that is available for general use in laboratory is a sophisticated optical instrument(light microscope) that can provide high-resolution images of a variety of specimens. Image quality is based largely on observer ability to use the microscope properly.

The light microscopy can be divided into six sub-categories:

-Bright field microscopy

-Dark field optics

-Ultraviolet microscopy

-Fluorescence microscopy

-Phase contrast microscopy

-Differential interference contrast (D.I.C).

Most microscopes in current use are known as compound microscopes, where a magnified image of an object is produced by the objective lens, and this image is magnified by a second lens system (the ocular or eyepiece) for viewing. Thus, final magnification of the microscope is dependent on the magnifying power of the objective times the magnifying power of the ocular. Objective magnification powers range from 4X to 100X. Lower magnification is impractical on a compound microscope stand because of spatial constraints with image correction and illumination. Higher magnification is impractical because of limitations in light gathering ability and shortness of working distances required for very strong lenses. Ocular magnification ranges are typically 8X-12X though 10X oculars are most common. As a result, a standard microscope will provide you with a final magnification range of ~40X up to ~1000X. Because the most frequently used objective lens is:

- 4x objective

- 10x objective

- 40x objective

-100x objective (oil immersion)

Before using a microscope, it is important to know the functions of each part:

·        Eyepiece Lens:  the lens at the top that you look through. They are usually 10X or 15X power.

·        Tube:  Connects the eyepiece to the objective lenses

·        Arm:  Supports the tube and connects it to the base

·        Base:  The bottom of the microscope, used for support

·        Illuminator:  A steady light source (110 volts) used in place of a mirror.  If your microscope has a mirror, it is used to reflect light from an external light source up through the bottom of the stage.

·        Stage:  The flat platform where you place your slides.  Stage clips hold the slides in place.  If your microscope has a mechanical stage, you will be able to move the slide around by turning two knobs.  One moves it left and right, the other moves it up and down.

·        RevolvingNosepiece or Turret:  This is the part that holds two or more objective lenses and can be rotated to easily change power.

·        Objective Lenses:  Usually you will find 3 or 4 objective lenses on a microscope.  They almost always consist of 4X, 10X, 40X and 100X powers.  When coupled with a 10X (most common) eyepiece lens, we get total magnifications of 40X (4X times 10X), 100X , 400X and 1000X.  To have good resolution at 1000X, you will need a relatively sophisticated microscope with an Abbe condenser.  The shortest lens is the lowest power, the longest one is the lens with the greatest power.  Lenses are color coded and if built to DIN standards are interchangeable between microscopes.  The high power objective lenses are retractable (i.e. 40XR).  This means that if they hit a slide, the end of the lens will push in (spring loaded) thereby protecting the lens and the slide.  All quality microscopes have achromatic, parcentered, parfocal lenses.

·        Rack Stop:  This is an adjustment that determines how close the objective lens can get to the slide.  It is set at the factory and keeps students from cranking the high power objective lens down into the slide and breaking things.  You would only need to adjust this if you were using very thin slides and you weren't able to focus on the specimen at high power. (Tip: If you are using thin slides and can't focus, rather than adjust the rack stop, place a clear glass slide under the original slide to raise it a bit higher)

·        Condenser Lens:  The purpose of the condenser lens is to focus the light onto the specimen.  Condenser lenses are most useful at the highest powers (400X and above).  Microscopes with in stage condenser lenses render a sharper image than those with no lens (at 400X).  If your microscope has a maximum power of 400X, you will get the maximum benefit by using a condenser lenses rated at 0.65 NA or greater.  0.65 NA condenser lenses may be mounted in the stage and work quite well.  A big advantage to a stage mounted lens is that there is one less focusing item to deal with.  If you go to 1000X then you should have a focusable condenser lens with an N.A. of 1.25 or greater.  Most 1000X microscopes use 1.25 Abbe condenser lens systems.  The Abbe condenser lens can be moved up and down.  It is set very close to the slide at 1000X and moved further away at the lower powers. 

·        Diaphragm or Iris:  Many microscopes have a rotating disk under the stage.  This diaphragm has different sized holes and is used to vary the intensity and size of the cone of light that is projected upward into the slide.  There is no set rule regarding which setting to use for a particular power.   Rather, the setting is a function of the transparency of the specimen, the degree of contrast you desire and the particular objective lens in use.

How to Focus Your Microscope

The proper way to focus a microscope is to start with the lowest power objective lens first, usually 4x and while looking from the side, crank the lens down as close to the specimen as possible without touching it.  Now, look through the eyepiece lens and focus upward only until the image is sharp.  If you can't get it in focus, repeat the process again.   Once the image is sharp with the low power lens, you should be able to simply click in the next power lens and do minor adjustments with the focus knob.  If your microscope has a fine focus adjustment, turning it a bit should be all that's necessary.  Continue with subsequent objective lenses and fine focus each time.

OBJECTIVES:

-to provide an experience in the use of microscope

-to illustrate the diversity of cells and microorganism

RESULTS:

Stained cell

Species: Staphylococcus aureus

Total magnification: 40X

Total magnification: 100X

Total magnification: 400X

The wet mount

Species: Saccharomyces cerevisiae

Total magnification: 1000X(oil immersion)

Species: Lactobacillus fermentum

Total magnification: 1000X (oil immersion)

DISCUSSION:

Stained Cell

Bacteria are stainso that they can be more easily visualized under the microscope. Some stains can also be used to identify and classify bacteria. Gram stain is a method of staining bacteria using a dye called crystal (gentian) violet. Gram's method helps distinguish between different types of bacteria species into two large groups.

The gram-staining characteristics of bacteria are denoted as Gram-positive or Gram-negative, depending upon whether the bacteria take up and retain the crystal violet stain or not. The Gram stain is almost always the first step in the identification of a bacterial organism, and is the default stain performed by laboratories over a sample when no specific culture is referred. A Gram positive results in a purple/blue color while a Gram negative results in a pink/red color.

Gram-positive bacteria retain the color of the crystal violet stain in the Gram stain. This is characteristic of bacteria that have a cell wall composed of a thick layer of a particular substance, specificallypeptidologlycan containing teichoic and lipoteichoic acid complexed to the peptidoglycan. Molecules of crystal violet combine with iodine molecules to form a complex within the microbial cell. The addition of decolorizer effectively dehydrates the peptidoglycan layer preventing the extraction of the crystal violet–iodine complex from the interior of the cell. The final step consists of the addition of safranin counterstain. The color from the retained crystal violet-iodine complex obscures the safranin counterstain to give the cells a dark blue, or purple, appearance via high resolution light microscopy.

The Gram-positive bacteria include staphylococci ("staph"), streptococci ("strep"), pneumococci, and the bacterium responsible for diphtheria (Cornynebacteriumdiphtheriae) and anthrax (Bacillus anthracis).

Gram-negative bacteria lose the crystal violet stain (and take the color of the red counterstain) in Gram's method of staining. This is characteristic of bacteria that have a cell wall composed of a thin layer of a particular substance (specifically, peptidoglycan covered by an outer membrane of lipoprotein and lipopolysaccharide containing endotoxin). Molecules of crystal violet combine with iodine molecules to form a complex within the microbial cell. The addition of Gram decolorizer effectively extracts the outer membrane and dehydrates the peptidoglycan layer. It is presumed that the inner membrane is extracted by the Gram decolorizer, but remains associated with the mureinsacculus . However, the thinner dehydrated peptidoglycan layer cannot prevent the extraction of the crystal violet–iodine complex from the interior of the cell through the peptidoglycan layer. The final step consists of the addition of safranin counterstain. As the crystal violet-iodine complex has been extracted from the cells, the safranin counterstain gives the cells a pink, or red, appearance via high resolution light microscopy.

The Gram-negative bacteria include most of the bacteria normally found in the gastrointestinal tract that can be responsible for disease as well as gonococci (venereal disease) and meningococci (bacterial meningitis). The organisms responsible for cholera and bubonic plague are Gram-negative

Staphylococcus aureus (S. aureus) is a bacterial species which appears as grape-like clusters when viewed through a microscope, and has large, round, golden-yellow colonies, often with hemolysis, when grown on blood agar plates.  S. aureus is a facultatively anaerobic, Gram-positive coccus. The bacteria are catalase-positive and oxidase-negative. S. aureus can grow at a temperature range of 15 to 45 degrees and at NaCl concentrations as high as 15 percent. Nearly all strains of S. aureus produce the enzyme coagulase: nearly all strains of S. epidermidis lack this enzyme. S. aureus should always be considered a potential pathogen; most strains of S. epidermidis are nonpathogenic and may even play a protective role in humans as normal flora. Staphylococcus epidermidis may be a pathogen in the hospital environment. Staphylococci are perfectly spherical cells about 1 micrometer in diameter. The staphylococci grow in clusters because the cells divide successively in three perpendicular planes with the sister cells remaining attached to one another following each successive division. Since the exact point of attachment of sister cells may not be within the divisional plane, and the cells may change position slightly while remaining attached, the result is formation of an irregular cluster of cells. S. aureus can cause a range of illnesses, from minor skin infections, such as pimples, impetigo, boils (furuncles), cellulitis folliculitis, carbuncles, scalded skin syndrome, and abscesses, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome (TSS), bacteremia, and sepsis. Its incidence ranges from skin, soft tissue, respiratory, bone, joint, endovascular to wound infections.

The wet mount

The wet mount method allow us to study the size and shapes of living microorganism as the slides were prepared at the time. Howeverthe mounting of specimens on microscope slides is often critical for successful viewing. Thus, the slides need to be carefully handled and take some precaution. In this experiment, Lactobacillus fermentum and Saccharomyces cerevisiaeslides were prepared. To put the coverslip, place one edge of it onto the slide and gently lowered it.Care must be taken to exclude air bubbles that would interfere with the viewing and hamper the organisms' movements. Then, the slides were observed under the microscope. The size and shapes of living microorganism only can be clearly seen using oil immersion objective lenswith totalmagnification of 1000X. It also enables we toobserve the motility of an organisms.

Conclusion

 As the conclusion,we able to learn the correct way to use a simple bright-field microscope correctly to the view prepared slides of microorganisms. Different species of microrganisms slides fromwhether the stained cells or wet mount was observed under different magnification from 4X up to 100X objective lens to examine the structure and size of microorganisms.

Reference

1)     Lab manual

2)    http://www.life.umd.edu/cbmg/faculty/wolniak/wolniakmicro.html

3)    http://www.microscope-microscope.org/basic/microscope-parts.htm

4)    http://ezinearticles.com/?Principles-of-Microscopes-and-Microscopy---Part-I&id=3284282

5)    http://en.wikipedia.org/wiki/Gram_staining

6)    http://en.wikipedia.org/wiki/Staphylococcus_aureus