Lab 2 ; Nur'Ain binti Zulkefli- 111399

LAB 2: MEASUREMENT AND COUNTING OF CELLS USING MICROSCOPE

2.1 Ocular Micrometer

   Introduction

     Ocular micrometer is use in order to measure and compare the size of prokaryotic and eukaryotic microorganism. Microorganisms are measured with an ocular micrometer which is inserted into the one of the microscope eyepieces. The micrometer, which serves as a scale or rule, is a flat circle of glass upon which are etched equally spaced divisions. This is not calibrated, and may be used at several magnifications. When placed in the eyepieces, the line superimposed certain distance markers on the microscope field. The actual distance superimposed may be calibrated using a stage micrometer on which parallel lines exactly 10 µm apart etched. By determining how many units of the ocular micrometer superimpose a known distance on the stage micrometer, you can calculate the exact distance each ocular each ocular division measures on the microscopic field. When you change objectives you must recalibrate the system. After calibration of ocular micrometer, the stage micrometer is replaced with a slide containing microorganism. The dimensions of the cells may then be determined.

   Objective

To measure and count cells using microscope

     Results

1)     Lactobacillus

    40 x magnifications

100 x magnifications

2)   Yeast

40 x magnifications

100 x magnifications

          Discussion

To compare and measure the size of prokaryotic and eukaryotic microorganisms, we used ocular micrometer. We use the lowest power objective to focus the microscope until the image on the stage micrometer is superimposed on the eyepiece scale and calibrate.

Prokaryotic and eukaryotic has different characteristic. Prokaryotic cell is a single cell while eukaryotic cell is more complex than prokaryotic and can be single or multicellular  cell. Prokaryotic cell has smaller size because prokaryotic does not contain many organelles compared to eukaryotic such as chloroplast, nucleus, endoplasmic reticulum and cell membrane.

The example of prokaryotic cell in this experiment is lactobacillus and yeast as the example of eukaryotic. During the experiment, we used  40x magnification and 100x magnification to observe the both cells.

	400x magnification		1000x magnification
calibrate	Stage micrometer	Ocular division	Stage micrometer	Ocular divison
	0.03 mm	38 division	0.09 mm	96 division
	7.9x10⁻⁴ mm	1 division	9.38x10⁻⁴ mm	1 division
	0.79 µm	1 division	0.94 µm	1 division

Size of microorganism :                                            2 division                                               5 division

YEAST :

400x magnification

       4 division x 0.79 µm

       =3.16 µm

1000x magnification

       7 division x 0.94 µm

       = 6.58 µm

LACTOBACILLUS :

400x magnification

       2 division x 0.79 µm

       =1.58 µm

1000x magnification

      5 division x 0.94 µm

      =4.7 µm




    Conclusion

As the conclusion, we are able to learn on measuring and counting the cells using microscope. An ocular micrometer is a glass disk that attaches to a microscope’s eyepiece. An ocular micrometer has a ruler; calibrated with a slide micrometer that allows the user to measure the size of magnified objects. The distance between the marks on the ruler depends upon the degree of magnification. We used this ocular micrometer to observe prokaryotic and eukaryotic cells since these two cells are very small.

Reference

http://wiki.answers.com/Q/What_is_ocular_micrometer

http://www.ehow.com/how_5019336_use-ocular-micrometer.html

Pearson International edition Biology 8th Edition, Campbell. Reece

2.2 Neubauer Chamber

Introduction

          Neubauer chambers are more convenient for counting microbes. The Neubauer is a heavy glass slide with two counting areas separated by a H-shaped trough (see figure 2.1). A special coverslip is placed over the counting areas and sits a precise distance above them.

Objective

To count cells using microscopes.

Result

Yeast 400x magnification

Discussion

          


          In the second experiment, Neubauer chambers are used for more accurate in counting microbes. In addition, the sterilization method is used to avoid contamination of microorganism. The types of sterilization that we used is flame sterilization and also Pasteur pipette to transfer the yeast to the slide.

          The chamber consists of 9 major large squares. In the calculation, we only use the middle of the large square.  Inside the large square, there contain another 16 smaller squares. Then choose 10 squares out of 16 squares to calculate the number of yeast.

56 ÷ 10 = 5.6

                 = 6

Volume = 0.05 x 0.05 x 0.01 mm

               = 2.5 x 10⁻⁵ mm

               = 2.5 x 10⁻⁷ ml

6 cells in 16 squares

1 square = 6 ÷16

                 = 0.375 cells in 2.5 x 10⁻⁷ ml  

1ml = 0.375 ÷ 2.5 x 10⁻⁷

       = 1500000 cells/ml

Conclusion

          As the conclusion, we can use two methods to measure and count cells using methods. The first one is by using ocular micrometer and the second one by using Neubauer chambers. By using Neubauer chambers, we can get more convenient in counting the cells compared to ocular micrometer. This is because, when using Neubauer chambers, the sterilization is needed to avoid any contaminant. Hence,  Neubauer chambers are suitable to count the cells.

Reference

  1.      Pearson International Edition Biology, 8^th Edition, Campbell, Reece

LAB2 : NURUL SAFFA BINTI ZAINAL ABIDIN 

EXP :2.1 OCULAR MICROMETER

INTRODUCTION 

Ocular micrometer is used in order to measure and compare the size of prokaryotic and eukaryotic microorganisms. Microorganisms are measured with an ocular micrometer which is inserted into the one of the microscope eyepiece. The micrometer, which serves as scale or rule, is flat circle of glass upon which are etched equally spaced divisions. This is not calibrated, and mat be used at several magnifications. When placed in the eyepiece, the line superimposed certain distance markers on the microscope field. The actual distance 10µm apart etched. By determining how many units of the ocular micrometer superimposed a known distance on stage micrometer, you can calculate the exact distance each ocular division measures on the microscopic field. When you change objectives you must recalibrate the system. After calibration of the ocular micrometer, the stage micrometer is replaced with a slide containing microorganisms. The dimensions of the cells may then be determined.

OBEJCTIVE 

To measure and count cell using a microscope

RESULT

1) Lactobacillus

400x magnification

1000x magnification

2) Yeast 

1000x magnication

400x magnification

DISCUSSION

From this experiment we can determined that yeast has bigger size compared to lactobacillus because yeast is the example of the eukaryotic organism while lactobacillus is example of prokaryotic organism. 

We observed both organism using 400x magnification and 1000x magnification(immersion oil) under the microscope due to it tiny structure.

In the first experiment we are using  ocular micrometer to measure and compare the size of prokaryotic and eukaryotic  microorganism. By using the lowest power objective, focus the microscope until the image on the stage micrometer is observed superimposed on the eyepiece scale and then calibrate :

	400x magnification		1000x magnification
calibrate	Stage micrometer	Ocular division	Stage micrometer	Ocular divison
	0.03 mm	38 division	0.09 mm	96 division
	7.9x10⁻⁴ mm	1 division	9.38x10⁻⁴ mm	1 division
	0.79 µm	1 division	0.94 µm	1 division

Size of microorganism :                2 division                                          5 division

YEAST :

400x magnification

       4 division x 0.79 µm

       =3.16 µm

1000x magnification

       7 division x 0.94 µm

       = 6.58 µm

LACTOBACILLUS :

400x magnification

       2 division x 0.79 µm

       =1.58 µm

1000x magnification

      5 division x 0.94 µm

      =4.7 µm

CONCLUSIONS

Eukaryotic microorganisms are more complex and can be multicellular or single cell. Prokaryotic microorganism are single cell. Usually prokaryotes organisms has smaller size compared to eukaryotes because it lacking in some organelles such as nucleus, cell membrane, endoplasmic reticulum and chloroplast. 

We also able to measure the size of microorganism by using ocular micrometer and then calibrate it.

REFRENCE

Pearson International edition Biology 8th Edition, Campbell. Reece

http://tami-port.suite101.com/prokaryotic-and-eukaryotic-cells-a32332

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

EXP : 2.2 NEUBAUER CHAMBER

INTRODUCTION

Neubauer chambers are more convenient for counting microbes. The Neubauer is heavy glass slide with two counting areas separated by a H-shaped (figure below). A special coverslip is placed over the counting areas and sits a precise distance above them.

OBJECTIVE

To counting  microbes by using Neubauer chamber

RESULT

                YEAST:

1000x magnification

DISSCUSION

For the second experiment we use Neubauer  chamber for counting microbes. There are some step of sterilization step we need to apply for this experiment which is we use flame sterilization to eliminate potential contaminants from exposed opening of the media bottle containing yeast during transfers. We also use sterile Pasteur pipette. All this to prevent us from infected and spread of microorganism.

The chambers contain many grids that producing 9 major large square. So for the calculation we only used middle large squares . Inside the middle large square there are 16 smaller squares. Then randomly choose 10 out of 16 smaller squares  and calculate the number of yeast cells in each of the squares and the cell concentration :

   56 ÷ 10 = 5.6

                 = 6

Volume = 0.05 x 0.05 x 0.01 mm

               = 2.5 x 10⁻⁵ mm

               = 2.5 x 10⁻⁷ ml

6 cells in 16 squares

1 square = 6 ÷16

                 = 0.375 cells in 2.5 x 10⁻⁷ ml  

1ml = 0.375 ÷ 2.5 x 10⁻⁷

       = 1500000 cells/ml

CONCLUSION

As a conclusion, we can count number of cell in colony and the cell concentration using Neubaeur chamber. sterilization process also applied in this experiment.

REFRENCE

Pearson International edition Biology 8th Edition, Campbell. Reece

http://www.ruf.rice.edu/~bioslabs/methods/microscopy/cellcounting.html

http://en.wikipedia.org/wiki/File:Neubauer_improved_counting_chamber.jpg

LaB 1~NuRaIn BiNtI aRzMi~111400~

INTRODUCTION

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

The wet mount method enables you to study the sizes and shapes of living microorganisms (drying or staining microorganisms distort them). It also enables you to determine if cell 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

1. Typical bacillus

40x magnification

100x magnification

400x magnification

2.  Lactobacillus fermentum

400x magnification

1000x magnification

3. Sacchamnyces arerisiae

400x magnification

1000x magnification

DISCUSSIONS

1.    Stained cells

From the observation of Typical bacillus we can classify it as the Gram positive bacteria. It has thick wall made of peptidoglycan and has rod-shaped. It can traps crystal violet in the cytoplasm and gives purple-black colour. The alcohol rinse does not remove the crystal violet, which mask red safranin dye is added.

            Bacteria cell walls that contain peptidoglycan, is a network of modified-sugar polymers cross-linked by short polypeptides. It used different magnification to observe the bacteria under microscope and to get the clear views, which are 40x, 100x, and 400x magnification.

2.   Wet mount

For these experiment, the wet mount methods which helps us to study about the shapes and the sizes of living organism. In this method, the drop of liquid of the specimen that is located between slide and cover glass is suspended. When using wet mount method, it is more fasters to prepared and it is possible to observe the living and moving organism. We observed the organism with different magnification that is 4x, 10x, and we also used immersion oil. The function of using the immersion oil is to get final resolution and brightness. We can get more clears image. In higher magnification, these characteristics are most critical.

We also used aseptic technique in this method. Inoculating wire is slowly heated until its glows orange. Cool it before attempting any bacteria. Then, we also heat the mouth of opening bottle. This is to make sure that we are not infected and to prevent the spread of microorganisms.

During this experiment, the Lactobacillus fermentum is observed under 40x, 100x magnification. We can see the rod-shaped structure and also the moving of organism. Lactobacillus cannot be seen under 4x and 10 magnification because it’s not tiny. It also same goes to yeast, we are unable to see the organism under 40x, 100x magnification. Yeast are the fungi which do not contain chlorophyl under the microscope. We can see it is cocci or spherical shape. We also can see clears images of shape when use oil immersion at 100x .                                        
                                                                  CONCLUSIONS

       There are two type of gram. Gram positive and gram negative. Gram positive and gram negative bacteria have similar internal but very different external structures. Gram positive bacterium has a thick, multi functions cell wall consisting mainly o f peptidoglycan surrounding the cytoplasmic membrane. Staining reaction take advantages of the facts that the cells or structure within the cell display dissimilar staining reactions that can be distinguish by the used of different type of dyes. Gram technique is used to differentiate two large groups of bacteria based on their different wall constituents. Gram positive bacteria stain violet due to the presence of the thick layer of peptidoglycan in their cell walls, which retains the crystal violet their cells are stained with.

        The immersion oil is used to take advantage of the highest resolving powers available to the microscopist. Unless this is a necessary requirement of the work in hand, stay with the lower power dry objectives. They are much less trouble to use and can provide magnification up to 600x (using a 40x, 0.65NA dry objective with 15x eyepiece) at quite acceptable resolution. Lactobacillus also has been identified as potential of probiotic.

Some samples can be placed directly under the microscope. However, many samples look better when placed in a drop of water on the microscope slide. This is known as “we mount”. The water helps to support the sample and it fills the space between the cover slip and the slides allowing light to pass easily through the slide of the sample, and cover slip.

REFERENCES

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

v http://www.micrographia.com/tutoria/micbasic/micbpt07/micb0700.htm

v http://www.mos.org/sln/sem/wetmount.html

v Pearson International Biology, 8th Edition, Campbell,Reece

Lab 1: Nurul Saffa Bt Zainal Abidin

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 method enables you to study the sizes and shapes of living microorganisms. It is 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

Typical bacillus

40x magnification

Typical bacillus

100x magnification

Typical bacillus

400x magnification

Lactobacillus fermentum

1000x magnification

Lactobacillus fermentum

400x magnification

Sacchamnyces arerisiae

1000x magnification

Sacchamnyces arerisiae 

400x magnification

DISCUSSION

1.  Stained cells

        By using technique called the Gram stain, we can classify it is Gram positive bacteria. It is rod shape and typical bacillus. Gram positive bacteria has a thick cell wall made of peptidoglycan that traps crystal violet in the cytoplasm. The masks the added red safranin dye. Most bacterial cell walls contain peptidoglycan, a network of modified-sugar polymers cross-linked by short polypeptides. So when we observe under microscope we use different magnification to get better view which is 4x, 10x and 40x.

2. Wet mount

                          Second experiment we use wet mount methods which helps us to study the sizes and shapes of living organisms. In wet mount, the specimen suspended in drop of liquid method, it is quick preparation and it is possible to observe living and moving organisms. We also observe the organisms using different magnification which is 40x,100x,400x and 1000x magnification. Immersion of oil is used to observed under 1000x magnification to get clearer view and brightness. This characteristics are most critical under higher magnification.

                           We also practice aseptic technique during the experiment. We should slowly heat the inoculating wire until it glow orange. Then, cool it before attempting any transfer of material. We also heat the mouth of an opening bottle. This is to make sure we are not get infection and prevent spread of microorganisms.

                    

                            During this experiment, we observe Lactobacillus fermentum under 400x and 1000x magnification. Thus we can see s rod-shaped structure and also the organisms is moving. Lactobacillus cannot be seen under the 40x and 100x magnification because it is too tiny to be seen. Same goes to yeast, we unable to see it under 40x and 100x magnification but can be seen under 400x and 1000x magnification. Yeast and fungi which do not contain chlorophyll. Under microscope we can see it is cocci or spherical shape. We can see clearer image of shape when use oil immersion at 1000x magnification.

CONCLUSION

         

                 Gram-positive bacteria ang Gram-negative bacteria. These staining reaction take advantages of the facts that the cells or structure within the cell display dissimilar staining reactions that can be distinguished by the used of different dyes. A gram positive bacteria can be identified by retaining a purple colour dye within its peptidoglycan exterior. This staining technique is used to help identify various bacteria. The gram positive bacteria that are purple hold stain due to it's layered cell membrane. It contain a peptidoglycan layer acts as a lattice trapping the crystal violet-iodine dye complex. Typical bacillus has a rod-shaped and the average bacillus is 0.5-1.0 um wide by 1.0-4.0 um long.

                    Many sample look better when use immersion oil. This is known as a wet mount. Oil helps support the sample and it fills the space between the cover slip and the slide allowing light to pass easily through the slide, the sample and cover slip. Lactobacillus fermentum has been identified as potential probiotic. The use of gut microbes as probiotics in food is aim towards preventing and treating various health disease. We also use wet mount method to see yeast clearly under microscope using 400x and 1000x magnification.

REFERENCES 

1. Pearson International Biology, 8th Edition, Campbell,Reece
2.http://en.wikipedia.org/wiki/Bacillus_(shape)
3.http://www.nutritionj.com/content/10/1/30
4. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC119863/