Practical

Dr. Waleed Khalid

Tues. 11/10/2015

PRACTICAL MICROBIOLOGY

LAB:1
Laboratory Safety Rules, Microscope and Preperation of smear

There are specific safety rules that are needed to be followed while working in microbiology lab. These safety rules include :

Wear a lab coat in lab.

Eating and drinking are forbidden in microbiology lab.
Thoroughly wash your hands with soap and water before and after lab.
Clean the lab bench with disinfectant before and after lab.
Dispose of all contaminated materials and slides in specific containers
Bacterial loop has to be sterilized by flame before and after use.

LIGHT MICROSCOPE

- A basic microscope consists of two lenses. The uppermost lens, called the ocular lens which is the part through which a person looks. The lower lens is the objective lens. Usually, several objective lenses are localized on a turret, allowing rapid changing of objective lenses. The eyepiece tube holds the ocular and objective lenses in place. Most microbiological specimens are mounted on glass slides and placed on the stage.


Procedure :
1 - Clean your lenses with lens paper.
2 - Set the microscope on the scanning (red lens (4*)).
3 - Focus using the coarse adjustment.
4 - Change to low power lens (yellow lens (10*)) and focus.
5 - Switch to high power lens (blue lens (40*)). Only use the fine adjustment knob.
6 - Switch the objective to half way between the high power and the oil immersion lens (black and white) (100*) .
7 - Place a drop of oil on the slide.
8 - Turn oil immersion lens into the oil.
9 - Check your image and only use fine to adjust.

PREPARATION OF SMEAR

Preparation of fixed smear :
From fluid material : ( such as broth culture , urine , sputum , pus , purulent exudates …, etc)
Sterilize the loop in benzene flame , and let it to cool.
Using a septic technique , withdraw a loopful of the specimen and spread it on the center of a clean slide to form a somewhat of 1-2 cm in diameter , then resterilize the loop
Allow the film to dry by air without heating.
The film is fixed on the slide by passing it 3 times through the benzene flame, allow the slide to cool before staining.

From solid material: ( such as a culture on agar i.e colonies )

Sterilize the loop in benzene flame and let it to cool.
Place a loopful of a clean water ( tap water can be used ) on the center of a clean slide.
By the resterilized loop, transfer a small portion of the colony to the drop of water, emulsify thoroughly and spread the mixture evenly on the slide to form a thin film of 1-2 cm of diameter
Dry and fix as mentioned above


AIM OF FIXATION:
Kill the microorganism
Make the microorganism stuck to the surface of the slide
Make the microorganism more permeable to the stain
Prevent the microorganism from going autolytic changes.

LAB: 2

STAINING METHODS
Simple staining technique:
Simple stains are used to demonstrate the presence of organisms and the nature of any cell present in the smear by applying only one dye.
Stains in general can be divided into three groups: Basic , Acidic and Neutral .

Acidic Stains

Basic Stains
Neutral Stains
Nigrosin
Crystal violet
Giemsa
Malachite green
Methylene blue
Leishman
Acid fuchsin
Safranin
Wright


Basic fuchsin

As bacterial cells are rich in nucleic acid ( which has a negative charge ) it will follow that “basic stain ,bearing its coloring matter in the positive cation , will be attracted to the organism and stain it” .Acid stain ,however, will not stain the bacteria ; they are used mainly for staining the background material a counterstaining color
Technique of simple staining:
1.flood the slide with loefflers methylene blue for 5-10min
2.wash off the stain with slowly running tap water
3.allow the slide to dry in air or placed it between two sheets of filter paper
4.examine under oil immersion

B.Differentional (compound) staining technique:

Staining technique consist of more than one dye used successively to identify organisms according to their type of reaction.
The most important examples for this group of stains are:
1.gram,s staining method
2.acid fast stain (zeihl neelsen methods)
3.spore stain

GRAM STAINING METHOD:

Is one of the most important methods widely used in bacteriology discovered in 1884 by gram (a Danish physician), using two dyes in sequence .each of different color. He found bacteria fall into two different categories:
A) Those that retained the first dye(crystal violet)throughout the staining procedure are known as “GRAM POSITIVE”
B) Those that lost the first dye (crystal violet)after washing with adecolorizing solution and stained with the second dye (safranine)are known as “GRAM NEGATIVE”


IN CONCLUSION, the gram positive bacteria appear violet ,while gram negative bacteria are red in color .therefore ,it is possible to differentiate between bacteria of the same morphology. furthermore, it can be used to determine the relative number and morphology of bacteria in a smear taken directly from a patient

PROCEDURE OF GRAM STAINING:

1-flood the slide with methyl e violet (crystal or gention violet),leave to act for 1-2min., wash with tap water
2-apply grams iodine (a mordant).leave to act for one minute. Wash with tap water.
3-apply 95%ethyl alcohol ( adecolorizer ), leave to act for 20-30 seconds ,wash with tap water.
4-apply saffranin (the counter stain).leave to act for 1-1.5min..wash with tap water, blot, dry in air and examine with oil immersion lens.

MECHANISM OF STAINING:

The division of bacteria into two categories, indicates abasic chemical differences between gram positive and gram negative bacteria. The most important differences are:
The cell wall of Gram-negative organisms have relatively little peptidoglycan and mainly consist of lipoproteins and polysaccharides. While in Gram positive organisms the peptidoglycan comprises the major part of the cell wall rendering them more rigid than Gram negative cells and less permeable for the dye iodine complex to diffuse freely out of the cell during the process of decolourization.
The more acid character of the protoplasm of Gram positive bacteria which is enhanced by treatment with iodine may partly explain their stronger retention of the basic dye.
Integrity of the cell wall.

LAB:3CULTURE MEDIA

Culture media are used for the recognition and identification of microorganism. The media are contained either in test tubes, plates (Petri dishes) flasks or screw capped bottles which must be thoroughly cleaned before use, then the medium and the container are subsequently sterilized by heat.

Common ingredients of culture media:

1. Peptone
2.Meat extract
3.Nacl
4. Agar agar a. Melting point 92-95 c
b. solidifing point 42-45 c
c. concentration 1.5-2%


Most pathogenic bacteria have restricted pH (7.2 – 7.4 ) . Therefore, pH of the media should be adjusted using 10% NaOH or HCL

TYPES OF CULTURE MEDIA :

- Culture media can be classified according to:
A - Physical state ( consistency ) of the media
1.Liquid (Fluid) media e.g. nutrient broth, peptone water, brain heart infusion. They are commonly used for primary cultivation.
2.Solid media e.g. nutrient agar ,blood agar, MacConkey's agar which are commonly used for cultivation of bacteria.
3. Semisolid media used for cultivation of spirochetes and to study motility. They contain 0.4 – 0.8 % of agar agar.

B. According to the use of media

1)Simple or basal media e.g. nutrient agar and nutrient broth.
They are used for the cultivation of common microorganism but not for the fastidious bacteria.

2)special-purpose media e.g. enriched, selective, differential, transport, sensitivity test, etc…

ENRICHED MEDIA :-

-Simple media enriched with appropriate substance ,e.g. Blood ,glucose ,serum and ascetic fluid ,most commonly used to cultivate fastidious microorganism like streptococci.

SELECTIVE MEDIA :-

-Containing inhibitory substance ( e.g. bile salt ,antibiotic, dyes…etc) which favour the growth of concerned microorganism and inhibit the growth of other . e.g. Maconkey's agar, Potassium tellurite agar, Bismuth sulphite agar , Deoxycholate citrate agar…etc


DIFFERENTIAL MEDIA
-Certain species produce characteristic growth that can be easily recognized, or can produce certain effects in the media e.g. hemolytic and non-hemolytic species on blood agar ,MacConkeys agar differentiates between lactose fermenter and non lactose fermenter gram negative bacilli.

Antibiotic susceptibility testing

This test is used to know the appropriate antimicrobial agents to be used for the treatment of the causative bacteria.
Disk diffusion test:
The culture media inoculated by the pathogenic bacteria and a paper discs impregnated by different type of antibiotic
(Each disc for specific antimicrobial agent eg disc for ampicillin and other for gentamicin, amikacin rifampicin …etc) are placed on this culture media. The culture is incubated at 37 C for 24-48 hr according to the type of mo. The test results are red as the followings:
A Zone of inhibition of growth (no growth around the disc) the bacteria is sensitive to this agent.
If the growth of mo reached the edge of the disc this mo is resistant to this antimicrobial drug.
LAB: 4
Culture of microorganisms from environment

- Microorganisms are found throughout the environment. For better study of these microorganisms pure culture is needed.Pure culture: a single kind of microorganism growing alone in a protect environment.There are two methods used widely for preparation of pure culture from mixed population.
1. steak plate method.2. pour plate method.
Streak plate method ( Quadrant plate)
This method is used for the isolation of pure culture of bacteria from mixed population e.g. sputum, urine, stool or from pus of infected wound or abscess.
Technique of Streak plate method :
Sterilize the loop in a benzene flame, cool it and streak the specimen over an area-A.
Re-sterilize the loop, cool it, then streak over an area-B from the distal part of area-A.
Continue the streak in the same manner for areas C and D.
Incubate the plates at 37°C for 24 hours.
A subculture is done after 24 hours incubation from one of the colonies needed for study on a sterile medium following the same technique mentioned previously. The culture produced is a pure one.


Pour plate method
-This method involves serial dilution of specimen and mixing with melted media. Then pour the content into sterile plate plates.

Cultural characteristics:

Cultural characteristics involve both :
1. Growth requirement.
2. Colonial morphology.
It is used to study the macroscopic characteristics of a pure culture on a solid medium.
Colonial morphology :
The following points have to be considered in describing a colony:
1. Size : measured by mm.
2. Shape : rhizoid ,circular, filamentous, irregular.
3. Elevation: flat, convex, raised, umbonate.
4. margin: entire lobulated ,filamentous
5. Consistency: dry, mucoid.
6. Surface texture: rough, smooth
7. Color or pigmentation.
8. Optical density : opaque, translucent, glistening.
9. changes in the inoculated medium e.g heamolysis.
10. Odour: bad or sweat musty odour.


Stock culture
Means preservation of a microorgansim in a culture medium for future study. It can be kept usually for 1 month at 4 °C.

Technique of stock culture:

Use a universal bottle containing medium in slanted position ( to provide a wide surface for inoculation and good nutrition).
Using a sterile loop inoculate the surface of the medium with one or few colonies of a pure culture.
Incubate the inoculated medium at 37 °C for 24 hours, then store at 4 °C.