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Script 7 All what we have learned so far is just a review, now we are going to concentrate on the bacteriology part. We are going to learn about the bacteria and it's shape that is called morphology, Colonies morphology are found when we grow the bacteria in the laboratory . The picture shows different shapes of different kinds of bacteria:

For example bacteria in chains which are couple of cells bonded together like bacilli rods in chains, and then you can see two of them together (doplococci) here you can see the spiral shaped bacteria, and you can see the cocci each 4 together(tetrad). And then you can have them in chains like streptococci like we mentioned the last time they all spherical in shape and in chains. Some times you can see that this chain gives specialty for bacteria which is bacteria meningitis or bacteria borellia. So we have different shapes of bacteria. Bacilli: also referred to as rods, may be short or long, thick or thin, and pointed or with curved or blunt ends, their shape is (rod) they could be pointed tapered at the end or they can be blunt or narrowed in the end .

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if it is long rods it could be mycobacterium . They may appear singly when you look at them under the microscope you will see them spread all over the slide or sometimes you can see each two together in pairs if its rod in shape we call them diplobacilli ,or sometimes you can see a couple of them in chains streptobacilli. You can see them also In long filaments or branched. Long filaments is a Property of some types of bacteria which are mycobacterium which cause Tuberculosis, and the way it's going to look under the microscope in a special stain(acid-fast) it's going to look filamentous. An average size of bacillus is 1*3 micrometer extremely short bacilli are called coccobacilli Short rods almost oval in shape. Examples of medically important bacilli pathogenic microorganisms that are important to us as human beings we are studying them a lot: 1- escherichia coli which is a short rod. 2-Clostridium which is small and oval in shape. 3- Proteus typical rod shape bacillus. Spiral (curved) shape bacteria: 1-Vibrio cholera it s a curved shape bacillus which cause Cholera has a comma shape

2-Campylobacter spp. Spiral in shape cause diarrhea like when you travel from county to another ex. From Jordan to Syria, and we call this travelers diarrhea. Helicobacter its one of campylobacter spp. Its very important Jordan its causes ulcer in stomach and if not treated when child till it's 30 or 40's it may lead to stomach cancer.

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3-Treponema spp 4-Borrelia spp. Both of them cause syphilis disease which is a sexually transmitted disease Borrelia is another kind of spiral bacteria that causes Lyme Disease which is spread in the north side of USA in the forest where there are many deer and the tick carries the disease, and it can be carried on dear and it will pass to the human and he will be infected and it may destroy all his arthritis, Chronic Arthritis.

5- Borrelia Hermsii these are the spiral shaped bacteria- in stained blood smear , a cause of relapsing fever. And there are many kinds of Borrelia.

We Have three categories of staining procedures:

1-simple stains: basic and acidic stain the same meaning of positive and negative stain so these are called simple stain and in this case you are going to be using one kind of stain. like crystal violet, pairopepsine, safronin. We do staining to the bacteria it self, or to the surrounding of the bacteria and the bacteria is going to appear as colorless and the background is colored, It depends on the kind of stain you are using.

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2-Structural staining procedure: Capsule stain: If you want to use the stain to see a the capsule structure on the bacteria such as Klebsiella pneumoniae. capsule: consist of polysaccharide a component outside of the cell it gives advantages to the cell like protection against antiphagocytic which means that this bacteria not going to be killed by phagocytic cells, because the anti genes will be covered inside the capsule, and they are not going to be appearing to phagocytic cells, the bacteria in this shape may disappear and it may proliferate in the body and avoid phagocytosis. *not all kind of bacteria have capsule. Spore stains: not all kind of bacteria have spores, only some of them, the bacteria that have spores are pathogenic like clostridium Bacilli species. They have advantage that the bacteria can live in a harsh environment and they can survive if they formed spores. Flagella stain: is also a special stain, the flagella can be present In certain type of bacteria not all of them and we are able to see them under the microscope if we used a special stain this stain will enhance the thickness of the flagella(increase the diameter)so we can be able to recognize or see this structure.

Simple bacterial staining technique: 1. Smear loopful of microbes onto slide . 2. Air-dry. 3. Drip methanol onto specimen to fix. 4. Flood slide with stain. 5. Rinse with water blot dry. 6.Examine with x100 objective .

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3-Differential staining: It's kind of a stain that can differentiate different structures in the same cell and we a.Gram stain. b. Acid-fast stan.

a.Gram Stain Procedure: Most bacteria are colorless, transparent and hard to see so the scientist developed a staining methods to examine bacteria. In 1883 dr. Hans Christian gram developed a staining technique The Gram stain OR Gram staining procedure, this Gram stain considered as the most important staining procedure because it differentiates between: 1-Gram positive bacteria . 2-Gram negative bacteria.

Procedure: 1-We make smear in the same way that we made it before in simple stain and fix it with heat.

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2- Add crystal violet stain and leave it for 1-2 minutes. 3- then we bend the slide and wash it with water until the color is gone. 4 we add mordant gram s iodine 2% and leave it for 1 minute mordant: substance that fix the stain. 5- we use 95 %alcohol or acetone alcohol it depends on what you have in the lab we keep adding drops of alcohol and let it for 5 seconds until purple color of crystal violet and iodine gone(decolorization step). 6-then we wash the smear again with water to remove all the excess of alcohol. (Now the gram negative bactria will be colorless or transparent and the gram postitive will be purple.) 7- add the other stain which is the red safranin and leave the stain for 1 minute. 8- wash the smear and let it dry to be able to see it under microscope. (at this stage the gram negative bactria will become red and the gram positive bactria will stay purple)

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The color of bacteria at the end of the procedure depends on their cell wall. if the bacteria were not decolorized they will be blue to purple from this we can conclude that the purple cells will be: y Gram positive because of its thick layer of peptidoglycan -11 layer- in their cell wall that will prevent or make it difficult to remove the crystal violet-iodine complex during the decolorization step(adding of alcohol or acetone alcohol) so it will stay purple. In the other hand the crystal violet was removed from some cells during decolorization step then the cells which were staind by the safranin , their color will be pink to red . we can conclude that red cells will be:

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y Gram Negative bacteria because it has thin layer (1 or 2 layers) of peptidoglycan in its cell wall which makes it easier to remove the crystal violet-iodine complex during decolorization and also make it easier for the safranin stain to enter the cell .

 Here is a very important table that shows the:

Lipopolysaccharides they considered the endotoxin in gram Negative bactria.

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o pictures for some Gram positive Bacteria:

They are both from the same genus streptococci but they are considerd a different species . Figure 4-22 : this chains of bacteria comes from a liquid culture lets say its streptococcous pyogens which cause pharyngities it will look purple in color Figure 4-23: Streptoccous pneuminiae which cause pneumonia its also a gram positive bacteria but it will appear in diplococci (we see it every two cells together) not in chains. Notes:  Baccilli and Streptococci can be both kinds of bacteria they can be a gram positive or gram negative bacteria.  Bacillus is one cell . Bacilli couple of cells together. Streptococcus is one cell. Streptococci is couple of cells together.

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Figure 4-24: from liquid culture it is called bacillus clostridium perfringens that causes gas gangrene its gram positive bacteria that has a rod shape , it also appear in purple.

Figure 4-25: it is also taken from liquid culture. clostridium tetani that cause tetanus its shape like a baseball bat it has a terminal spores , this bacteria is very significant microorganisim because it might kill you, also it can form sopres this spores can cause death even if they were dead . it can be used as a biological weapon because spores can spread easily and easy to inhale.

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Figure 4-26: Gram positive bacteria that can live inside or outside epithelial cells (blood stream), it can t survive it might be killed outside the cell so this bacteria enter the cell .

o Pictures for some Gram negative Bacteria(red color) Figure 4-28: Borrelia burgdorferi is spiral shaped bacteria which cause lyme disease. Both of them are gram negative bacteria because they are red .

b. acid-fast stain : its considered both special and differential stain to distinguish the Gram Variable bacteria  Gram Variable bacteria: Bacteria that can t be distinguished by Gram staining because when we stain them some of them will appear as purple and others as red on the same slide but they are actually the same kind of bacteria. Like: Mycobacterium spp.

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Mycobacterium spp. Are identified using acid-Fast stain procedure in this procedure carbol fuchsin( bright red dye) is driven to bacteria cell using heat. The heat is necessary because the cell wall of mycobacterium contain waxes and lipid that called mycolic acid which prevent the stain from entering the cell so the heat softens the wax so the stain will be able to penetrate. It also going to kill the micro organisms, second it's going to fix the micro organisms on the slide, and once you heat the Carbol Fuchsin on the top of the sample, you are opening pores on the cell wall of mycobacterium and the stain will enter to the cell, and after you remove the heat the stain will trap inside the cell wall and the it's going to be stained. If we use acid alcohol(decolorizing agent), it won't remove the stain, that's why it's named acid-fast stain, because you can't remove the stain, because you forced the stain to get in.

This stain has another modification we can use Phenol with Carbol Fuchsin, in this case we don't have to use the heat in order to force the penetration of the stain.

Done by :
y y

gewanna ghazal amal sliman

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