Microbiology

The hanging drop technique requires a few extra steps compare to the wet mount technique. The hanging drop technique is a more appropriate way to observe bacteria motility since the cover slip has a grove or a space for the bacteria liquid to be put on. After a few procedures, the bacteria liquid is observed using a compound microscope. Under a 10X magnification, there are no bacteria are visible except for a few particles.  The magnification is added until it was 40X. The image of bacteria is seen with less clear view because of the resolution.

There is a way to observe movement in bacteria. It is called the motility test. In this test, the motility media is stab with inoculating needle. This test is done to differentiate species of bacteria that are motile. The media and reagent used contains tryptose, sodium chloride, agar and a color indicator. There will be growth going out from the stab line if the bacteria are motile. If there is no motility, there will be no growth along the stab line.

There are two types of movement observed. The first movement is random and zipping across the field of sight. This is possible with the assistant of an appendage called the flagella. The flagella act as a propeller for bacteria to move in a rotation fashion, gliding and forward fashion. There are bacteria with single flagella and there are with numerous flagella disperse all around it. The example of motile bacteria is Salmonella typhymurium and E. coli.

There is another kind of movement observed under the microscope. This movement is different from the flagellated movement. It is not because of the bacteria itself rather it is the result of the bombardment of the bacteria by water molecule. This will create movement due to capillary action or convention current. It is the main problem when dealing with classification and identification of bacteria. Dead cells and nonliving particle tend to have this movement due from the phenomena stated. To overcome this problem, the motility test can be done( as stated above).

The second method is the simple wet mount technique. Under the 40X magnification, there is no movement or motility observed. This is because the bacteria are rendered motionless because the slit is pressed upon it. However, it is a weak hypothesis.

The hanging drop technique giver positive result whereas the simple wet drop technique gives no result. There are several factors that affect the outcome of the experiment. The main factor is the flaw in preparing the slide. The slit is not properly inserted. The bacteria solution is not properly administered. Some of the bacteria are dead but still shows movement

The haging drop technique is the most suitable method to observe motile bacteria.

Microbiology Lab 1

Discussion Practical 1

The microscope is an instrument used to see objects that are too small for the naked eye. It is critical if we want to observe and study microbiological life form. Microscope can be categorized into two types - the light microscope and the electron microscope. The light microscope is suitable to observe the organism on a cellular scale whilst the Electron microscope is suitable for sub-cellular (inside cell) scale. The light microscope can be divided further into bright field (stains) microscopy, dark field microscopy, phase contrast microscopy, Differential interference contrast microscopy, confocal microscopy and florescent microscopy.  

Compound microscope is made up from different components, parts and sections. The critical parts are the objectives lens and the confocal lens for magnification. The image from the objective lens is magnified again by the ocular lens. The total magnification is the product of objective lens magnification times with the ocular lens magnification. There are 4X magnification, 10X magnification, 40X magnification and 100X magnification on the objective lenses. The ocular lens magnification is fixed at 10 times. The compound microscope uses the light to make the object visible.

Compound microscope consisted of:-

• 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 used in place of a mirror. 
• Stage:  The flat platform where you place your slides.  Stage clips hold the slides in place. The stage 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.
• Revolving Nosepiece or Turret:  This is the part that holds two or more objective lenses and can be rotated to easily change power.
• 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.

The final image observed from the compound microscope is inverted and magnified depending on the lenses used. The letter ‘e’ appeared upside down. When the stage is moved to the left, the image ‘e’ is shifting to the right and vice versa. When the stage is moved away from the observer, the image ‘e’ moved down and vice versa.

The letter "e" is observed with a higher magnification lens. The fine texture of the paper becoming more visible as the objective lens is changed from low to high. The letter "e" is now missing from the field of view, since the specimen is too enlarged to see all of it at one time. This explains why it is important to carefully center small specimens within the field of view, before changing lenses. The higher the magnification of the objective lens, the smaller the field of view. On high magnification(100X), the oil immersion technique is used. Application of 100X would result in refraction of light as it passes through the glass slide and air. Immersion oil have the same refraction index with the glass(objective lens) so it can avoid the refraction of light. The image of the specimen can be seen clearly. Oil immersion objectives are used only at very large magnifications that require high resolving power. Objectives with high power magnification have short focal lengths, facilitating the use of oil. The oil is applied to the specimen (conventional microscope), and the stage is raised, immersing the objective in oil. The oil will minimize the lost of light path from refraction.

The microscope is handled with care. The utilization log must be recorded upon usage. The microscope is cleaned and stored after use

Discussion Practical 2

There are about 2400 of genus Salmonella. Almost all the members are potentially pathogenic. Salmonella lives in the intestinal tract of many animals including human. Under unsanitary conditions, they can contaminate food. The ability to contaminated food makes it a gram-negative type. Salmonellae are Gram-negative , facultative anaerobic bacteria of the family Enterobacteriaceae. They utilise citrate as a sole carbon source and generally ferment glucose but not sucrose or lactose. Salmonella enterica serovar typhimurium is the among the most common Salmonella causing infections in the human intestine. In humans, it can causes diarrhea, fever, and abdominal cramps 12 to 72 hours after infection and may last for up to 7 days.

Under the microscope, the Salmonella typhimurium are seen as color pink. During the staining process, the crystal-violet smear is washed away by the iodine making it colorless again. It turns into pink when the counter stain Safranin is added. Gram-negative bacteria are difficult to destroy and cannot be killed by antibiotic. It is because the composition of the cell wall Peptidoglycan. Under a high magnification(100X), the oil immersion technique is used(please see Discussion 1). Although the image is a bit dark because of the resolving power, the bacteria is still distinctive. The bacteria appeared to be rod-like. The image is drawn on a piece of paper.

Because of the extreme small size, the bacteria are observed under a very strict procedure. This help in the discipline of Microbiology and improved the use of certain technique and a way to handle microscope.