BACTERIAL STAINING (GRAM - POSITIVE AND GRAM – NEGATIVE

BACTERIAL
INTRODUCTION
Most bacteria are classified into two broad categories: Gram negatively stained cell
positive and Gram negative. These categories are based on
their cell wall composition and reaction to the Gram stain test.

The Gram staining method, developed by Hans Christian Gram, positively stained cells
identifies bacteria based upon the reaction of their cell walls to
certain dyes and chemicals. Differential Staining
Gram staining: procedure to subdivide bacteria by their reaction
Staining is used in a variety of ways in order to color the to the stain to:
background of a cell, discern types of cells and to discern a. Gram positive (+) retains the color
structures of a cell. b. Gram negative (-) decolorized.
o The simple stain can be used as a quick and easy way Uses 4 solutions:
to determine cell shape, size and arrangements of • Basic dye: methylene blue, nigrosine, Indian ink
bacteria. • Mordant dye: iodine
• Decolorizing agent: alcohol, acetone
Gram staining is a type of differential stain that works by • Counter stain: safranin
distinguishing gram positive and gram negative cells by coloring
them violet or red, respectively. Procedure of gram staining
o Staining is used in a variety of ways to color the 1. Prepare smear on clean slide.
background of a cell, discern types of cells and to 2. Stain with crystal violet (30 secs) – primary stain
discern structures of a cell. A differential stain is when 3. Rinse with water
multiple dyes are used to stain a cell that take 4. Flood the film with gram iodine and allow it to act for 30
advantage of chemical differences in a cell. secs. - mordant
5. Rinse with water
ADVANTAGES 6. Decolorized with 95% alcohol. - decolorizer
1. Provide contrast between microorganisms and their 7. Rinse with water
backgrounds, permitting differentiation among various 8. Counter stain with safranin for (20-30 secs) – counter
morphology types. stain
2. Permits study to internal structures of bacterial cell, such 9. Rinse with water and blot dry.
as cell wall, vacuoles or nuclear bodies and other 10. Examine under oil immersion objective.
cellular structures.
3. Enables the bacteriology to use higher magnification. Acid-fast staining: differentiate two types of gram positive cells:
those that have waxy mycolic acids in their cell walls, and those
Staining technique that do not.
• Staining is a technique used to enhance contrast in Two methods:
samples, generally at the microscopic level. 1. Ziehl-neelsen technique
• Chemical or physical union between dye and like 2. Kinyoun technique
component of the cell. Used to correct identification of Both use carbolfuchsin as the primary stain
microorganism.
Preparation of acid-fast staining
Simple staining 1. Prepare smear on clean slide.
Method using a single stain (dyes such as crystal violet, 2. Carbon fuchsin (3-5 mins) - primary stain
methylene blue and carbon fuchsin) to color a bacterial 3. Apply heat until steam, rises (Ziehl neelsen) - mordant
organism provides a quick and easy way to determine cell 4. Rinse with water
shape, size, and arrangement. 5. Decolorized with 95% alcohol (15-20 secs) - decolorizer
1. A bacterial smear is stained with methylene blue for 6. Rinse with water
one minute. 7. Counter stain with methylene blue (2 mins) – counter
2. Stain is briefly washed off slide with water. stain
3. Water drops are carefully blotted off slide with bibulous 8. Rinse with water and do not allow to dry
paper. 9. Examine under oil immersion objective.

Before staining it is essential to fix the bacterial sample to o the Notes:

slide. Smear is prepared in the following way: • 30 seconds to 2 mins staining time only
1. With a wire loop place a small of the broth culture or a • LPO - HPO - Oil Immersion
loop full of bacteria on a clean slide. • Cationic dyes - are dyes that can be dissociated into
2. A drop of water over it. positively charged ions in aqueous solution.
3. Spread the culture to form a thin film. • Anionic dyes - are dye molecule dissociate into
4. Allow slide to dry in the air or by holding it above a negatively charged ions in an aqueous solution.
Bunsen flame. • The Gram staining process includes four basic steps,
5. Avoid excess heating. including:
1. Applying a primary stain (crystal violet).
2. Adding a mordant (Gram's iodine).
3. Rapid decolorization with ethanol, acetone, or a
mixture of both.
4. Counterstaining with safranin.
• Acid-Fast Staining - to determine if the bacteria is
infected and has a high content.
• Non-Acid Fast - will stain blue or green with
counterstaining.
• Carbol Fuchsin - the primary stain used in acid fast
staining.
• Two common bacteria that produces endospores:
1.Genus Bacillus (an obligate aerobe often living in the
soil)
2.Genus Clostridium (an obligate anaerobe living in the
gastrointestinal tract of animals)
There are three components of specimen quality are: Labels should include the patient's name, distinct
(a)proper specimen selection (i.e., the appropriate hospital identification number, and hospital
type of specimen must be presented), room number, as well as the name of the requesting
(b) proper specimen collecting, and doctor, the culture site, and the date and
(c) proper specimen transit to the laboratory. time of collection.
When clinical specimens are gathered and handled Laboratory requests should include the following
incorrectly, the following may occur: information:
(a)The etiologic agent (causative agent) may not be the patient's name, age, gender, and unique hospital
detected or killed identification number; the name of the
(b) indigenous microflora overgrowth may mask the requesting clinician; specific information about the
infection, and/or type of specimen and the site from which it
(c) contaminants may obstruct pathogen identification was collected; the date and time of collection; the
and the diagnosis of the patient's infectious initials of the person who collected the
disease by the lab/physician specimen; and information about any antimicrobial
Clinical specimens are numerous types of specimens agent(s) that the patient is receiving.
collected from patients and used to diagnose Adequate clinical information should always be
or follow the progression of infectious diseases, such as provided to the laboratory to aid in the
blood, urine, feces, and cerebrospinal fluid. performance of relevant analyses.
The nurse’s responsibility is either in the collection, Such as: laboratory request accompanying a wound
transport, instruction, correct site selection (ex. specimen should not simply state "wound";
In the doctor’s order for a sterile urine analysis and rather, it should state the specific type of wound (e.g.,
others). burn wound, surgical site infection), the
The following general precautions should be observed anatomical site, or on left or right side.
when collecting clinical specimens for lab:
Specimen Selection:
• Specimen must be carefully chosen for the suspected COLLECTION AND TRANSPORTATION OF CLINICAL BIOLOGICAL
infectious disease's (usually based om SPECIMEN
doctor’s order).
Specimen Collection, • The laboratory diagnosis of an infectious disease begins with
• gathered in a way that eliminates or reduces the collection of a clinical specimen for examination or
contamination with indigenous microorganisms. processing in the laboratory (the right one, collected at the right
• gathered where contamination is least likely to occur. time, transported in the right way to the right laboratory).
• if possible, specimens should be acquired prior to start • Proper collection of an appropriate clinical specimen is the first
of antimicrobial medication. If not, step in obtaining an accurate laboratory diagnosis of an
informed lab antimicrobial agent(s) that the patient is infectious disease.
receiving. • Guidelines for the collection and transportation of specimens
• Most specimens should be collected during acute should be made available to clinicians in a lucidly written format.
stage of disease, while some viruses, during • The guidelines must emphasize two important aspects:
prodromal stage. - Collection of the specimen before the administration of
• performed with care and tact to avoid harming antimicrobial agents.
patient, causing discomfort, or causing undue - Prevention of contamination of the specimen with externally
embarrassment. present organisms or normal flora of the body.

• If patient is to collect the specimen, he must be given Collection and transportation of specimens
clear and detailed collection instructions.  Apply strict aseptic techniques throughout the
• sufficient quantity of specimen must be obtained. procedure
• should be placed or collected into a sterile container  Wash hands before and after the collection.
(types of collection devices and specimen  Collect the specimen at the appropriate phase of
containers specified per institution) disease.
• When feasible, utilize sterile, disposable specimen  Make certain that the specimen is representative of the
containers. infectious process (e.g. sputum is the specimen for
Specimen Transportation pneumonia and not saliva) and is adequate in quantity
• protect from heat and cold for the desired tests to be performed.
• brought to lab as soon as possible  Collect or place the specimen aseptically in a sterile
• If delayed, some delicate pathogens might die and/or appropriate container.
•some specimens must be delivered to the lab on ice,  Ensure that the outside of the specimen container
whilst others should never be refrigerated clean and uncontaminated.
or placed on ice due to the fragile and sensitive nature  Close the container tightly so that its contents do not
of the pathogens. leak during transportation.
• obligate anaerobes die when exposed to air, they  Label and date the container appropriately and
must be kept oxygen- complete the requisition form.
free during transport to the lab.  Arrange for immediate transportation of the specimen
• pathogens may be overgrown, inhibited, or killed by to the laboratory.
any indigenous microflora in the material.
• Specimens should be collected and brought to the Criteria for rejection of specimens
laboratory as early in the day as possible to Criteria should be developed by a laboratory on the basis of
allow lab professionals enough time to process the which the processing of a specimen may not be done by the
material, especially if the hospital or clinic does not laboratory. The following are some examples:
have 24-hour lab service.  Missing or inadequate identification.
• must be handled with great care so that patients,  Insufficient quantity.
couriers, and healthcare professionals are  Specimen collected in an inappropriate container.
not contaminated. ( Ex. Patient is in home or isolation  Contamination suspected.
area).  Inappropriate transport or storage
- Specimens must be placed in a sealed plastic bag  Unknown time delay.
and transported to the laboratory  Hemolyzed blood sample.
immediately and carefully. (On a case-by-case basis).
The specimen container must be correctly labeled, Nursing Functions for Specimen Collection
and it must be accompanied by an 1. Explain procedure, gain client's participation
authorized laboratory test requisition with adequate 2. Collect right amt. of specimen at the right time
instructions. 3. Place specimen in correct container
4. Label container accurately (addressograph), plastic bag
Collection of specimens
Blood
 Whole blood is required for bacteriological
examination.
 Serum separated from blood is used for serological
techniques.
 Skin antisepsis is extremely important at the time of
collection of the sample.
 Tincture of iodine (1-2%), povidone iodine (10%) and
chlorhexidine (0.5% in 70% alcohol) are ideal agents.
However, some individuals may be hypersensitive to
iodine present in some of these.
Sputum is processed in the laboratory for etiological investigation
While collecting blood for culture, the following points must be
of bacterial and fungal infections of the lower respiratory tract.it
remembered:
is of utmost importance in the diagnosis of pulmonary
 Collect blood during the early stages of disease since
tuberculosis.
the number of bacteria in blood is higher in the acute
 Select a good wide-mouthed sputum container. which
and early stages of disease.
is preferably disposable, made of clear chin plas leak
 Collect blood during paroxysm (uncontrollable) of fever
proof material
since the number of bacteria is higher at high
 Give the patient a sputum container with the
temperatures in patients with fever.
laboratory serial number written on it. Show the patient
 In the absence of antibiotic administration, 99% Culture
how to open and close the container and explain the
positivity can be seen with three. cultures.
importance of not rubbing off the number written on
 Small children usually have higher number of bacteria
the side of the container.
in their blood as compared to adults and hence less
 instruct the patient to inhale deeply 2-3 times, cough
quantity of blood needs to be collected from them.
up deeply from the chest and spit in the sputum
container by bringing it closer to the mouth.
 Make sure the sputum sample is of good quality. A
good sputum sample is thick, purulent and sufficient in
amount (2-3 mi)

Urine collection
 Whenever possible, the first urine passed by the patient
at the beginning of che day should be sent for
examination, This specimen is che most concentrated.
 Midstream urine (MSU) for microbiological examination
\ is collected as follows:
Cerebrospinal fluid (CSE)  Female patients Mash the bands Cleanse the area
Examination of CSF is an essential step in the diagnosis of any around the urethral opening which dean water, do the
patient with evidence of meningeal irritation or affected area with a sterile gauze pad, and collect the urine
cerebrum. watch the labia held apart
 Almost 3-10 ml of CSF is collected and part of it is used  Male patients Wash due hands before collecting a
for biochemical, immunological microscopic specimen ( middle of the urine flow)
examination and remaining and for bacteriological or
fungal examination. Time of Specimen Collection
• Best time to collect urine and sputum is early in the morning
when patient wakes up (because microorganisms have had the
opportunity to multiply for several hours)
• For collection of blood for culture best time is when the
temperature of patient is rising (because at that time maximum
number of microorganisms are present in the blood)

Stool
 Faecal specimens for the aetiological diagnosis of
acute infectious diarrheas should be collected in the
early stage of illness and prior to treatment with
antimicrobials.
 The feces specimen should not be contaminated with
The following important precautions need to be taken for CSF
urine.
collection and transportations.
 Collect the specimen during the early phase of the
• Collect CSF antimicrobial therapy before is started.
disease and as far as possible before the administration
• Collect CSF in a screw - capped sterile container and not in an
of antimicrobial agents.
injection vial with cotton plug.
 1 to 2 gm quantity is sufficient. If possible, submit more
• Do not delay transport and laboratory investigations.
than one specimen on different days.
• Transport in a transport medium if delay in processing is
 The fresh stool specimen must be received within 1-2
unavoidable.
hours of passage.
• CSF is a precious specimen, handle it carefully and
 Store at 2-8°C
economically. It may not be possible to get a repeat specimen.
 Most pathogens will survive for up 48 hours at room
- Perform collection physical and inspection indicate findings
temperature. Specimens are unacceptable if medium
immediately after on laboratory requisition form.
is held for more than one week or there is detectable
- Store at 37°C, if delay in processing is inevitable.
drying of the specimen.

Throat swab
 Depress the tongue with a tongue blade
 Swab the inflamed area of the throat, pharynx, or
tonsils with a sterile swab taking care to collect the DUS
or piece of membrane.
 Transport in sterile transport tube
Bone marrow is collected by a doctor who is well trained in this
procedure.
 Decontaminate the skin overlying the site from where
specimen is to be collected with 70% alcohol followed
by 296 tincture of iodine.
 Aspirate 1 ml or more of bone marrow by sterile
percutaneous aspiration.
 Collect in a sterile screwcap tube
 Send to laboratory immediately.

Rectal swab
 Insert swab at least 2.5 cm beyond the anal sphincter
so that it enters the rectum.
 Rotate it once before withdrawing.
 Transport in Cary and Blair or other transport medium

Transportation of specimens
• Specimens to be sent to other laboratories require special
attention for safe packing of the material.
• Guidelines authorities are and usually the same issued by
national should be strictly followed.
• For hand-carried transportation over a short distance, the
specimen should be placed upright in appropriate racks

For long distance transportation. It should be placed in three

containers.
 A primary container which has the specimen and is
leakproof with a screw-cap
 A secondary container which is durable, waterproof
and made of metal or plastic with a screw-cap. It
should have enough absorptive material to absorb the
contents of the primary container should the latter
break or leak. On its outside, the details of the
specimen should be pasted.
 A tertiary container is usually made of wood or
cardboard. It should be capable withstanding the
shocks and trauma of transportation. Dry ice can be
kept between this and the secondary container along
with sufficient absorbents and provision for the escape
of carbon dioxide to prevent a pressure build-up inside.

• In general, most specimens should be processed in the

laboratory within 1 to 2 hours after collection.
• In practice, a 2-to 4-hour time limit is probably more practical
during a normal working day.