Question

Lab Practical I – Microscopy, Aseptic technique, Bacterial culturing/selection, and Staining:

Briefly describe the most salient points for each section. Remember, if you know why the

assay is performed, how it works, and how to interpret it you will do very well on the final.

Section 3-1 and 3-5: Microscopy, Simple stains, and Smears

Section 1-3 and 1-4: Streaking plates, Transfer, and Colony Selection

Section 3-7: Gram Staining

Section 3-12, 3-13, and 5-28: Motility (Microscope-wet mount), Flagella Staining, and Motility

test

Section 3-8: Acid Fast Staining

Section 3-9, and 3-10: Capsule and Endospore Staining

Answer 1

lab practical I-

Microscopy:- Microbiology microscopes are used to identify and visualize some tricky samples including bacteria, algae and fungi. Power objective lenses with high resolution as well as observation techniques such as phase contrast microscopy and darkfield microscopy are key for observing some of earth's smallest creatures.... Dark field microscopy done to see Treponema Pallidium.

The following types of microscopes are in use now.
• Bright-field or light microscope
• Dark field microscope
• Phase contrast microscope

Fluorescence microscope
• Electron microscope....

Aseptic technique:- Aseptic technique is a standard healthcare practice that helps prevent the transfer of germs to or from an open wound and other susceptible areas on a patient’s body...

Aseptic technique helps prevent healthcare-associated infections (HCAIs). An HCAI is an infection that a person acquires as a result of treatment from a healthcare professional...

Common HCAIs include:

• catheter-associated urinary tract infection
• central line-associated bloodstream infection
• Clostridium difficile infection
• surgical site infection
• ventilator-associated pneumonia

there are several different aspects of aseptic technique practices:

• barriers :-

  Barriers

  Barriers prevent the transfer of germs between healthcare professionals, patients, and the environment. Aseptic barriers include:

• sterile gloves
• sterile gowns
• sterile masks
• sterile drapes
• protective wrappers on sterilized instruments

• patient and equipment preparation:-
• disinfecting a patient’s skin using antiseptic wipes
• sterilizing equipment and instruments before a procedure
• keeping sterilized instruments inside plastic wrappers to prevent contamination before use

• environmental controls:-

  Maintaining an aseptic field involves:

• keeping doors closed
• minimizing movement in and out of the aseptic field
• limiting entry to necessary personnel only
• permitting only one patient per aseptic field

• contact guidelines:-

Bacterial Culturing:- Culture media are required to isolate die bacteria from the clinical specimens; following which the appropriate biochemical tests can be performed 10 identify the
causative agent.

TYPES OF CULTURE MEDIA
Bacteriological culture media can be classified in two ways.
A. Based on consi~tency, culture media are grouped into:
1. Liquid media (or broth)
2. Semisolid media
3. Solid media
B. Based on die growth requirements, culture media are
classified as:
1. Routine laboratory media: They are prepared from
nutrients, such as aqueous extract of meat, peptone,
etc. 11iey can furdier be classified into various types
based on functional use or application, as follows-
• Simple/ basal media :- tey contain minimum ingredients that support the
growth of non-fastidious bacteria. Examples include-
• Pep1onewa1er: It contairis peptone (I%)+ NaCl (0.5%)
+ water
• Nutrient broth: ll is made up of peptone water+ meat
extract (I%). ll is available in three forms: (1) meat
extract, (2) meat i,uusion, (3) meat digest brodi.
• Nutrient agar: It is made up of nucrient broth+ 2% agar  
• Semisolid medium: ll is prepared by reducing die
concentration of agar to 0.2 to 0.5 %.

• Enriched media :-  When a basal medium is added with additional nutrients,
such as blood, serum or egg. it is called enriched medium.
In addition to non-fastidious organisms, they also support
the growth of fastidious nutritionally exacting bacteria.
Examples include: Blood agar, Chocolate agar, loefflers serum slope...

• Enrichment broth:- lliey are the liquid media added widi some inhibitory
agents which selectively allow certain organism to grow
and inhibit others. 11tis is important for isolation of the
padwgens from clinical specimens whicli also contain
norma.l Hora (e.g; stool and sputum specimen). E.xamples
for enrichment broth include:
• Tetradiionate broth-used for Salmo,iel/aTyphi
• Gram- negative broth- used for isolation of Sltigella
• Selenite F broth- used for isolation of Shigella
• Alkaline pepwne water (APW)- used for Vibriocltolerae

• Selective media :- Selective Media
lliey are solid media containing inhibitory substances that
inhibit die normal Oora present in the specimen and allow
the pathogens to grow.
• Lowenstein-Jensen (LJ) medium: It is used for isolation
of Mycobacteriwn tuberculosis.
• 1liiosulphate citrate bile sail sucrose (TCBS) agar: ll is
used for isolation of Vibrio species .
• DCA (deoxycholate citrate agar): It is used for die
isolation of enteric padiogens, such as Salmonella and
Shigella from stool .
• XLD (i.ylose lysine deoxycholate) agar: It is also used for
same purpose 85 diat DCA.
• Potassium tellurite agar (PTA): It i5 is used for isolation
of Corynebacterium diphtlteria.e.
• wilson Blair bismuth sulfite medium: It is used for
isolation of St,lmonella Typhi.

Differential media
These media differentiate between 1wo groups of bacteria
by using an indicator, which changes the color of the
colonies of a particular group of bacteria but no1 the other
group.
• MacConkey agar: ll is a differential and low selective
medium, commonly used for the isolation of enteric
gram-negative bacteria .
It differentiates organisms imo LF or lactose
fennenters (produce pink colored colonies, e.g.
Fsc/1ericl1ia colt) and NLF or non-laccose fermenters
(produce colorless colonies, e.g. Slligella).
Composition: ll contains pep1one, lactose, agar,
neu1ral red (indicator) and 1aurochola1e.
Most laboratories use combination of blood agar
and MacConkey agar for routine bacterial culture.
• CLED agar (cyslelne lactose electrolyle-deflcienl
agar): lliis is another differential medium similar 10
MacConkey agar. capable of differe11tiating between LF
and NLF. It is used as an alternative to combination of
blood agar and MacConkey agar, for the processing of
urine specimens .
Advantages over MacConkey agar: ll is less inhibitory
than MacConkey agar, supports the growth of gram-
positive bacteria (except~ hemolytic Streptococcus)
and Candida.
Advantage over blood agar: IL can prevent the
swarming of Proteus.
Anaerobic Culture Media
Anaerobic media contain reducing substances which
take-up oxygen and create lower redox potential and
thus permit the growth of obligate anaerobes, such as
Clostridium. Examples are as follows:
• Robertson's cooked meal (RCM) broth: It contai11s
chopped meat particles (beef heart), whicl1 provide
glutathione (a sul01ydryl group containing reducing
substance) and unsaturated fany acids. I! is the most
widely used anaerobic culture medium. ll is
also used for maintenance of stock cultures.
• Other anaerobic media include:
• lhioglycolla1e brod1
• Anaerobic blood agar
BHIS agar (Brain-hear! infusion agar) with
supplements ( vitamin K and he min)
Neomycin blood agar
• Egg yolk agar
Phenyl ediyl agar
Ba.cteroides bile esculin agar (BBE agar)
Defined or Synthetic Media
Chemically defined media are used for various
experimental purposes. lliey are prepared exclusively
from pure chemical substances in such a way that dieir
composi1io11, i.e exact quantity of each chemical used is
known.
Simple Synthetic Media
lliey contain a carbon and energy source, such as glucose,
or lactose, and an inorganic source of nitrogen, usually
in the form of ammonium chloride, phosphate or sulfate
and various inorganic salts in a buffered aqueous solution.
lliey provide the basic essentials for the growth of many
non-fastidious he1ermrophs, bu! they will not support the
growth of fastidious bacteria.
Complex Synthetic Med/a
Here. in addition to die simple synthetic media certain
aminoacids, purines, pyrimidines, and other growth
factors are incorporated. Hence, they can also support the
growth of more exacting bacteria......

staining:- Structural details of bacteria cannot be seen under light
microscope due to lack of contrast. Hence, ii is necessary
to use staining methods to produce color concras1 and
thereby increase 1he visibility. Before staining, die fixation
oft he smear to die slide is done.
Fixation
Fixation is the process by which the internal and ext ernal
structures o f cells are preserved and fixed in position. It also
inactivates the enzymes that might d isrupt cell morphology.
It toughens cell structure so that they do not change during
staining. It kills.and fixes the cell.son to the slide.
There are two types of fixation as follows:
• Heat fixation: It is usually done for bacterial smears by
gently flame heating an air-dried film of bacteria. This
adequately preserves overall morphology but not structures
w ithin the cells.
• Chemkal fixation: It can be done using ethano~ acetic
acid, mercuric chloride, formaldehyde, methanol and
glutarald ehyde. They are used to protect the fine internal
structure of cells. This is useful for examination of bJood
smears.
llie fixed smear is stained by appropriate staining
technique.
COMMON STAINING TECHNIQUES USED IN
MICROBIOLOGY
• Simple stain: Basic dyes, such as mediylene blue or
basic fuchsin are used as simple stains. 11iey provide
the color contrast, but impart die same color to all die
bacteria in a smear.
• Negali,•e staining: A drop of bacterial suspension is
mixed with dyes, such as India ink or nigrosin. llie
background gets stained black where as unstained
bacterial/yeast capsule stand ou1 in contrast. ll1is is
very useful in the demonstration of bacterial/yeast
capsules which do not take up simple stains.
• Impregnation methods: Bacterial cells and s1ruc1ures
1ha1 are 100 diin to be seen under die light microscope,
are diickened by impregnation of silver salts on their
surface to make diem visible, e.g. for demonstration of
bacterial Oagella and spirochetes.
• Differential slain: Here, two stains are used which
impart different colors 10 different bacteria or bacterial
structures, which help in differentiating bacteria. llie
most commonly employed differential stains are-
• Gram stain: II differentiates bacteria into gram-
positive and gram-negative groups
• Acid-fast stain: It differemia1es bacteria into acid-
fast and non acid-fast groups
• Albert stain: It dlfferentiates bacteria having meta-
chromatic granules from other bactera that do not
have.

note :- dear student i have answered the lab 1 part but due to such long question and constarint of time i couldnt amswer all... Please repost the question seperately. ..

also due to some error there is a lot spelling msitake u may come across please pardon me