MICROSCOPES

MicroscopesMicroscope : an instruments used

to examine very small objects (Specimens) in microbiology.

Functions :To allow us to study the morphology

of bacteria (size, shape, arrangement, motility and staining reaction )

Types of microscopes

1-light microscope 2-Dark field microscope 3-fluorescent microscope4-Phase contrast microscope5-Electron microscope

•Light Microscope

•Eye piece•Objective lenses•Stage•Diaphragm •Condenser•Light source•Adjustment knobs

STAINING METHODS

Types of stain1-Simple stain (one dye is used )Methylene blue, safranin, crystal violet.

2-differential stains( two dyes are used)Gram stain ,Ziehl-Neelsen stain.

3-special stainsfontana stain.

4- Fluorescent stained antibodyAntibodies for surface antigens of bacteria

carrying a fluorescent dye

Simple stains 1- preparation of

smear:• Take a loopfull of bacterial

suspension and spread it onto an area of about 1 cm squaresquare

• Leave to dry in air or at the Leave to dry in air or at the warmth of a Bunzen flamewarmth of a Bunzen flame

• Fix the smear by: Fix the smear by: a- passing it into a flame three timesa- passing it into a flame three times

b- dipping into ethyl alcoholb- dipping into ethyl alcohol Fixation leads to coagulation of proteins and fixation on the surface of slide.

Simple stains 2-staining of the smear:a- Flood the smear with several drops of a stain like methylene blue and leave for 2 min.

b- wash the stain gently with water

c- Dry the smear by leaving it in air for a few minutes or by blotting with a filter paper.

Simple stains

3- Examination under the microscope•Put a drop of oil onto the stained smear•Examine the smear under the oil immersion lens•Describe what you can see under the microscope

Chains of bacillui

Tetrads of cocciChains of beads

Bunches of cocci

Gram-stain• Principles: • Bacteria differ from one another chemically

and physically and may react differently to stains.

• Bacteria are divided into1- Gram-positive bacteria (violet in colour)2- And Gram-negative bacteria (red in colour)

Gram-stain:Steps of staining:• 1- Prepare a bacterial smear and fix it

• 2- Flood with crystal violet (primary stain) and leave 60 sec. Wash gently with tap water.

• 3- flood with iodine solution (mordant) and leave for 2 min and then wash gently with tap water.

Gram-stain:Steps of staining:4-Declourize with drop by drop 95% ethanol alcohol (decolourization of Gram-negative bacteria), till the washing becomes faint purple (this takes about 20 sec). Wash gently with water.

5- Flood the smear with safranin (counter stain) and leave for 2 min.

6- Dry the smear by blotting, add a drop of oil and examine under oil immersion lens

Technique of isolation of separate colonies of

bacteria

Technique for isolates• Sterilize the loop by heating to

redness on the top of a flame.

• Take one loop-full of bacteria and streak the surface of nutrient agar plate to spread bacteria in area [1] as indicated in the photo.

• Sterlilize the loop and make other streaks starting from the side of area [1] as shown in the photo to spread bacteria in area [2].

• Repeat the same procedure to spread bacteria in area [3].

Colonies isolated

Types of media :

Types of media :1-basal media2-Enriched media3-Selective media4- Enrichment media 5-Differential media6- selective and indicator media

1-Basal media

1- Nutrient broth2-Peptone water (Water soluble

products obtained from protein materials digested by proteolytic enzyme as trypsin and pepsin).

3- Nutrient agar4-Nutrient gelatin nutrient agar

2-Enriched media

Useful for fastidious organisms

Prepared by Addition of blood, yeast extract, brain heart infusion

Examples:1-Blood agar2-Chocolate agar

3-Selective mediaContain substances that inhibit the

growth of some organism but have no effect on the organism of test .these inhibitors may be dyes ,chemicals or antibiotics.

An example is Mannitol salt agar used to separate staphylococcus aureus

Mannitol salt agarStaphylococcus aureus produce yellow colouration

4- Enrichment mediaThese are liquid media with

selective properties enhance the multiplication of organisms.

As:1-Selenite broth.2-Alkaline peptone water3-Tetrathionate broth medium

5-Differential media Contain some substances that

are changed visibly as a result of metabolic activity of organism as:

1-Sugar media 2-Triple sugar iron agar (TSI)

6- Selective and indicator media .

Composed of selective and indicator supplements in basal media as:

1- MacConkey’s agar2-Deoxycholate citrate agar (DCA).3-TCBS(Thiosulphate citrate and

bile salt (selective) sucrose (test sugar) and bromothymol blue (pH indicator)

MacConkey agar