To understand the basic principles of microscope, and use of microscope To learn and prepare bacterial smears from NB and NA; spreading, fixation and staining of specimen To become familiar with simple and differential staining, and use of basic dyes to stain cell wall of cells. To see Brownian motion and flagella movement and differentiate them To learn usage of depression slide To learn temporary-wet mount technique and hanging drop technique Be familiar with some stains and know the difference between the basic and acidic stains To be familiar with the techniques used to observe living cells under microscope
Procedure:
Main Points of protocols
The Use of Microscope
1. Turn on microscope, firstly use 4x lenses and then up to 40x to magnify image of specimen 2. take clean slides and label them as following B.Subtilis àCV P.vulgaris àHDT (use depression slide) S.aureus àMB S.cerevisiaeàTWM E.coli àS
First we cleaned the objectives of the microscope with cleaning solution and cleaned the benches with zephiran. And we prepared 3 bacterial smears as following the precedure below: Smear Preparation
1. Gently shake culture of cells if you are preparing from NB 2. Remove a loopful of culture aseptically 3. Spread evenly to middle 2/3 of slide 4. If you prepare from NA, then put a drop of dH2O to middle of slide 5. Then aseptically take a small colony from plate 6. Mix it with dH2O 7. Pass to fixation procedure
Fixation of specimen to slide
1. Wait for air dry for 2-3 minutes 2. Perform heat fixation 3-4 times over flame 3. Be careful about over heating, cool it with your hand by touching
Simple Staining
1. Apply 2-3 drops of stain to cover the smear as indicated below B.Subtilis àCrystal Violet S.aureus àMethylene Blue E.coli àSafranine 2. Wait for action of dye for 1 min 3. Rinse with water (do not apply directly), remove excess dye 4. Blot dry with tissue paper to suck excess water
Observation under Microscope 1. To see Brownian motion and movement of bacteria, take clean slides and label as S.cerevisiaeàTWM P.vulgaris àHDT (use depression slide) 2. Drop one drop of yeast suspension over the TWM slide and touch one edge of coverslip to the drop and lower coverslip slowly 3. For Hanging drop technique, use depression slide and by using Vaseline form a barrier around edge of cover slip 4. Perform a drop of NB culture of P.vulgaris to middle of cover slip and turn depression slide upside-down 5. Fix the coverslip and slide 6. Now put under the microscope and observe at 100X by applying oil over slide.
Results & Observations
S.aureusà Methylene Blue E.coli àSafranine B.SubtilisàCrystal V. àcoccus àrod àrod àblue-purple àpurple àvioloet
In this experiment, we have learned several basic molecular biology techniques and use of equipments such as smear preparation (staining, fixation, observing under microscope) and microscope respectively.
There are two types of classification according to grams staining; positive and negative depending on whether the bacteria take up the crystal violet stain or not so gives purple color or not. Positive gram bacteria have a kind of cell wall composed of a thick layer of peptidoglycan so it keeps crystal violet inside. On the other hand, gram negative bacteria lose stain and stained with counter stain and gives pink-red color of safranin. Character of cell wall of this kind of bacteia is a thin layer of peptidoglycan.
There are several steps in our experiments. For example, we did fixation to inactivate enzymes found, to harden the structure of smear and stich the smear to glass. I want to note that smear is the thin film of bacteria suspention on the slide.
To see slides under the microscope we used sometimes 100X magnification therefore we used oil to get clear images and tried to collect light by use of ligt because light is not scattered by using oil.
We stained several kinds of bacteria such as B.subtilis, S.aureus, and E.coli with Crystal violet, Mehylene Blue and Safranin respectively. Results of staining are given in “results and observation” part but I want to emphasize that we could determine relative size of this bacteria. We found that B.subtilis is thinner and longer than others and both B.subtilis and E.coli have rod shape structure but E.coli is more spherical and bigger than S.aureus and smaller than B.subtilis. On the other hand, S.aureus was the smallest one and it was coccus bacteria (spherical shape). After simple staining procedure, we saw that S.aureus was in blue-purple color, B.subtilis is in violet color and finally we saw purple staining of E.coli by using safranin.
Moreover, we also tried to see Brownian motion and flagellar motion of bacteria. For this purpose, we used Hanging drop technique with P.vulgaris species to see flagellar motion and S.cerevisiae (yeast suspension) to see Brownian motion of cells. The hanging drop and wet mount techniques allow for observation of living organisms. The wet mount tends to dry out quickly under the heat of the microscope light; it is simpler to perform than the wet mount, but it is useful for short-term observation only. In the results part I tried to show this but I want to indicate that we saw colorless cells in the WMT and you should care about looking immediately after the specimen prepared with WMT because cell suspension dries and Brownian motion stops rapidly. This motion is a random motion without help of any organ like flagella. It depends on mainly kinetic energy of any cell and concentration differences of them around. In the hanging drop technique we used a special kind of slide; depression slide by covering around of the hollow with Vaseline and prevent escape of bacteria with their flagellar motion. We could see flagellar motion of P.vulgaris under the 100X magnification, because they have long flagella to move fast. Their movements are in general straight to a point and then they return to other direction; however some of them make a kind of motion they turn like a propeller. These techniques are usually performed without the addition of any stains; therefore, the organisms can be difficult to see.
There are several things to take care of while smear preparations. Too thin smear is a problem in the case of smear preparation from NB cultures. This occurs when cells taken NB culture is not dense, or cells are taken from upper part and less dense part of it without shaking, or heat fixations not enough to fix the cells on the slide. On the other hand, there may be too thick smear in the case of smear preparation from NA cultures. In this case, e should try to take less amount of culture from agar because a thick smear may prevent proper light penetration so lessening visibility, and difficult to discriminate individual cells. Finally, cells might be destroyed if heat fixation occurs more than expected so we can see false results, we should also be careful about proper heat fixation.
I can tell something about microscope and its parts and functions; The eyepiece magnifies the image, usually 10X. The low-power objective further magnifies the image, up to 4X. The high-power objectives further magnify the image, from 10Xto 40X. The nosepiece holds the objectives and can be turned to change from one objective to another. The body tube maintains the correct distance between the eyepiece and the objectives. This is usually about 25 cm, the normal distance for reading and viewing objects with the naked eye. The coarse adjustment moves the body tube up and down in large increments to allow gross positioning and focusing of the objective lens. The fine adjustment moves the body tube slightly to bring the image into sharp focus. The stage supports a slide. The stage clips secure the slide in position for viewing. The diaphragm located under the stage, controls the amount of light that is allowed to pass through the object being viewed. The light source provides light for viewing the image. It can be either a light reflected with a mirror or an incandescent light from a small lamp. The arm supports the body tube. The base supports the microscope.
In conclusion, these exercises were successful and they were a good and essential part of study and learn microbiology. New procedures were practiced, and further understanding of simple staining, smear preparation, and observation of cells both living and nonliving(fixations) under microscope were gained.
