ADDIS ABABA UNIVERSITY

COLLEGE OF HEALTH SCIENCES

DEPARTMENT OF MEDICAL LABORATORY SCIENCES

Assessment of Gram stain competency and associated factors among laboratory

professional in clinical laboratories at Mekelle city health facility, Mekelle, Ethiopia

By:

Fikadu Miruts

Advisors: FatumaHassen (BA,BSc,MPH, PhD candidate)

HabtamuMolla (Bsc, Msc, PhD candidate)

A thesissubmitted to the Department of Medical Laboratory Sciences, College of Health

Science, Addis Ababa University, in partial fulfillment of Master of Science Degree in
Clinical Laboratory Sciences (Laboratory management and Quality Assurance Specialty
Track)

May, 2020

Addis Ababa, Ethiopia

 ADDIS ABABA UNIVERSITY

COLLEGE OF HEALTH SCIENCES

DEPARTMENT OF MEDICAL LABORATORY SCIENCES

Assessment of Gram stain competency and associated factors among

laboratory professional in clinical laboratories at Mekelle city health
facility

By:

Fikadu Miruts

Signed by the Examining Committee

External Examiner _____________________________ Signature__________ Date_______

Internal Examiner________________________________ Signature_________ Date_______

Advisor ________________________________________Signature_________Date________

Advisor _________________________________________Signature__________Date_______
_________________________________ ________________ ________________
Chairperson of the Department Signature Date
Name of the principal investigator By: Fikadu Miruts

Department of Medical Laboratory Sciences

College of Health Sciences, Addis Ababa
University

Advisors
Fatuma Hassen (MPH, PhD candidate)
Habtamu Molla (Msc, PhD candidate)

Department of Medical Laboratory Sciences

College of Health Sciences , Addis Ababa
University
Tel: +251 911 418062…Fatuma Hassen
Tel: +251910722375….Habtamu Molla

Full Title of the Project Assessment of Gram stain competency and

associated factors among laboratory
professional in clinical laboratories at
Mekelle city health facility

Type of protocol Medical

Duration of the project Four month

Total cost of the project 23,006.5 Birr
Address of the PI Email: [email protected]
Tel:+251914210892
Acknowledgment
First of all I would like to thank to my advisors, Habtamu Molla (Lecturer, Addis Abeba
University) and Fatuma Hassen (Lecturer, Addis Abeba University) for their unreserved
scientific guidance, comments, and support starting from topic selection to the development
of thesis.

I would like also to thank to Mekelle University College of Health Science Library for
providing the Internet service for searching the relevant literatures to this study.

I would like also to thank Microbiology team especially Kibra Hailu and Letekidan Lemlem
for their assistance during panel slide preparation and next I would like to thank to haftay
haileslase for guiding how to implement my thesis

I
Abstract

Background:Gram stains are initially used as a pre-analytical indicator of specimen quality and
acceptability for culture. It also gives the clinician preliminary information regarding the nature of
potential pathogens present in thepatient specimen and thus serve to guide empirical therapy. This
procedure is still considered a high-complexity procedure by the Clinical Laboratory Improvement
Amendments (CLIA) program. The manual nature of the staining process and the subjectivity of
Gram stain interpretation contribute to the incidence of errors.

Objective:Assessment of Gram stain competency and associated factors among laboratory

professional in clinical laboratories at Mekelle city health facility

Methodology: A cross-sectional study was conducted from January 2020 to April 2020 on 77
different health facilities present at Mekelle city. Convenient sampling method was used to
collectData from 148participants; of those are 81, 62 and 5 from private, governmental and uniformed
health facility respectively.Qualitative data were collected from medical laboratory professional
through a structured self-administered questionnaire and panel slide at on-site assessment of their
performance. Question and panel slide was used to assess knowledge and skill of laboratory
professionals respectively. Panel slides were prepared from American Type Culture
Collection(ATCC)by using known bacterial strains and no organism slides were prepared from patient
sample.Bloom’s cut off point of59 % or below as low, 60-80 % as medium and above 80: as high was
used to describe the knowledge and practical skills of the respondents.

Result:Among 155 eligible medical laboratory professionals, 148 (95.5%) of them participatedand
most of them were males 78(52.7%). Onehundred twenty seven 127(85.2%), Nineteen 19(12.8%) and
2(1.3%) participants scored low, medium and high skill level respectively. The level of skills and
knowledge of medical laboratory professional had significant associated witheducational level,
SLIPTA levelof health facility,training, higher institution type and sex had significant association
withstudy participant about gram stain (P < 0.05).

Conclusion:The present study showed that the majority of medical laboratory professional had low
knowledge and skill in gram stain examinations. Attention should be given to develop training
strategies that can improve laboratory professional knowledge and skill level. This could be achieved
through pre service and in service training and also giving adequate emphasis to gram stain related
practical training, continuous followupand Regular competence assessment (supervision).

Key words:

Professional competence, clinical performance, Gram Stain

II
Table of content

Table of Contents
Acknowledgment …………………………………………………………………..…………………..I
Abstract………………………………………………………………………………………………...II
Table of contents………………………………………………………………………………………………………………………..……III

List of tables…………………………………………………………………………………………....V

List of Abbreviation…………………………………………………………………………………...VI
1. Introduction ........................................................................................................................................ 1
1.1Background…………………………………………………………………………………..……...1

1.2 Statement of the problem……………………………………………………………………….3

1.3 Significance of the study………………………………………………………………………..5
2. Literature review……………………………………………………………………………………6
3. Objective .......................................................................................................................................... 9
3.1. General objective: ........................................................................................................................ 9
3.2. Specific objectives: ...................................................................................................................... 9
4. Materials and methods ...................................................................................................................... 10
4.1. Study Area ................................................................................................................................. 10
4.2. Study Design and Period ............................................................................................................ 10
4.3 Population ................................................................................................................................... 10
4.3.1 Source population ................................................................................................................ 10
4.3.2 Study population .................................................................................................................. 11
4.4. Eligibility criteria ....................................................................................................................... 11
4.5. Study variables ........................................................................................................................... 11
4.5.1. Dependent variables: ........................................................................................................... 11
4.5.2. Independent variables: ........................................................................................................ 11
4.6 Sample Size Determination and Sampling Techniques ............................................................ 12
4.6.1 Sample Size Determination .................................................................................................. 12
4.7. Measurement and Data collection .............................................................................................. 13
4.7.1Data collection tools and procedure ...................................................................................... 13
4.7.2 Panel Slide Preparation and Distribution ............................................................................. 14
4.8 Data management and Data Quality Assurance:...................................................................... 14
4.9 Data analysis and presentation: ................................................................................................. 15
4.10 Operational Definitions:............................................................................................................ 16

III
 4.11 Ethical considerations ............................................................................................................... 17
4.12 Dissemination of the result ....................................................................................................... 17
5.Result………………………………………………………………………………………………..18
5.1 Socio-demographic characteristics of the study participant……………………………........……18
5.2 Resource access of study participants……………………………………………………20

5.3Knowledge of medical laboratory professional in mekelle city health facility……………...…...21

5.4 Response of medical laboratory professional on each theoretical knowledge question about Gram
stain..…….………………………………..…………………………………………………………...23
5.5 The level of the skills of the medical laboratory professional on gram stain examination……….24

5.6 Errors of Medical Laboratory Professionals on gram stain examination………………………… 26

6.Discussion……………………………………………………………………...……………………27
7.Limitation……………………………………………………………………………………………30
8.Conclusion…………………………………………………………………………………………..30
9.Recommendation……………………………………………………………………………………31
10.Reference…………………………………………………………………………………………..32
11. Annexes……………………………………………………………………………………………35
Annex I: Information Sheet (English Version)……………………………………………………..35
Annex II: Informed Consent form (English Version)………………………………………………37
Annex III: Data collection tool for assessment of gram stain competency and associated factors
among laboratory personnel in clinical laboratory at Mekelle city health facility…………………38
Part 1: socio demographic data……………………………………………………………….38
Part 2: Gram stained panel slides report form for gram stain competency assessment……..….39
Part 3: Competence assessment for theoretical bases in Gram staining for medical laboratory
professionals working in Mekell city health institute, Mekelle, Ethiopia.....................................40
Annex IV: Media preparation for blood agar……………………………………………………….42
Annex V: Gram stain smear preparation procedure………………………………………………...43
Annex VI: Gram Stain Procedure…………………………………………………………………..44
አባሪ(Annex) VII : የስምምነትቅጽ(Consent form) የአማርኛውቅጂ(Amharic version)……………………..45
አባሪ( Annex)VIII: የመረጃቅጽ(Information Sheet) የአማርኛውቅጂ(Amharic Version)………………….46
Annex IX: Declaration……………………………………………………………………………...48

IV
List of tables

Table 1: Socio demographic characteristics of the study participant in Mekelle city health
facility, Tigray, Ethiopia…………………………………………………………………...19

Table 2: Resource access of study participants at Mekelle city health facility, Tigray, Ethiopia
2020………………………………………………………………………………………….20

Table 3: Knowledge of medical laboratory professional related to background characteristics

in mekelle city health facility, Tigray, Ethiopia, 2020 (n=148)………………………….…22

Table 4: Participants’ knowledge about Gram stain among medical laboratory professionals
at Mekelle city, Tigray, Ethiopia, 2020 (n= 148)……………………………………………23

Table 5: Skill of medical laboratory professional on each slides about Gram stain…………24

Table 6: The level of the skills of the medical laboratory professional on gram stain
examination in Mekelle city health facility, Tigray, Ethiopia, 2020……….…………..……25

Table 7: The level of Errors of Medical Laboratory Professionals on gram stain examination
at Mekelle city health facility, Tigray, Ethiopia 2020…………………………………..…..26

V
List of Abbreviation and Acronym
AAU = Addis Ababa University
ATCC= American Type Culture Collection
ACSH= Ayder Comprehensive Specialized Hospital
CAP = College of American Pathologists
CLIA = Clinical Laboratory Improvement Amendment
CPD = Continuing Professional Development
DRERC= Departmental Research and Ethics Review Committee
LPTP = Laboratory Proficiency Testing Program
PI = Principal Investigator
PT = Proficiency Test
QC = Quality Control
SLMTA = Strengthening Laboratory Management Towards Accreditation
SLIPTA = Stepwise Laboratory Improvement Process Towards Accreditation
U.S = United States

VI
1. Introduction
1.1 Back ground

Clinical laboratory is a laboratory where a tests are carried out on clinical specimens to get
information about the health of a patient to help in diagnosis, treatment, and prevention of
disease. Clinical Medical laboratories are an example of applied science, as opposed
to research laboratories that focus on basic science, such as found in
some academic institutions(1).

Laboratory quality can be defined as accuracy, reliability and timeliness of reported test result
so, the results must be as accurate as possible, all aspects of the laboratory operations must be
reliable, and reporting must be timely,this is important in a clinical or public health setting.
When making measurements, there is always some level of inaccuracy. The challenge is to
reduce the level of inaccuracy as much as possible, given the limitations of our testing
systems. An accuracy level of 99% is acceptable, but the resulting 1% error can become quite
large in a system where many events occur, such as laboratory testing. If inaccurate results
are provided, there are different consequences those are: unnecessary treatment,treatment
complications, failure to provide the proper treatment, delay in correct diagnosis, additional
and unnecessary diagnostic testing. So, these consequences result in increased cost in time
and personnel effort, and often in poor patient outcomes. (2).

Clinical microbiology laboratory testing plays animportant role in the detection, diagnosis,
and treatment of infectious diseases and public health disease surveillance. Microbiology
laboratories are often the first lines of defense in the detection of antibiotic resistance and in
the identification of outbreaksof, e.g., food-borne infection, and are responsible for reporting
certain infectious diseases to public health authorities. Therefore, it iscritical to ensure high-
quality testing and results that are accurate and precise (3).

Gram staining is one of the laboratory test methods which is named after the Danish
bacteriologist Hans Christian Gram who originally devised it in 1882 and published in 1884
to discriminate between Pneumococci and Klebsiellapneumoniaebacteria in lung tissue.Gram
stain is a classic biological protocol that is still actively used to differentiate bacteria into two
possible classifications: Gram-positive cells, in which the primary stain is retained and Gram-
negative cells, in which the primary stain is lost (4, 5, 6 ).

1
Gram stains are initially used as a pre-analytical indicator of specimen quality and
acceptability for culture. They also give the clinician preliminary information regarding the
nature of potential pathogens present in the patient specimen and thus serve to guide
empirical therapy. Although the Gram stain has been the staple of clinical microbiology
laboratories for over a century, it is still considered a high-complexity procedure by the
Clinical Laboratory Improvement Amendments (CLIA) program. The manual nature of the
staining process and the subjectivity of Gram stain interpretation contribute to the incidence
of errors (7, 8, 9, 10 ).Error on specimen sampling,Specimen processing, smear preparation,
and prior antibiotictherapy are all factors that can have an adverse impact onGram stain
result.

Gram stains remain the cornerstone of diagnostic testing in the microbiology laboratory for
the guidance of empirical treatmentbefore to availability of culture results.Misinterpretation
of Gram stains may adversely impact patient care, and yet there areno comprehensive studies
that have evaluated the reliability of the technique and there are no established standards for
performance.In this study, clinical microbiology laboratories at four major tertiary medical
care centers evaluated Gram stain errorrates across all non-blood specimen types by using
standardized criteria (11).

In Ethiopia, most medical laboratories in health care facilities-cannot perform microbial

culture for the diagnosis of microorganisms. Microbial culture for the diagnosis of
microorganisms is a limited service. This was due to lack of skilled-man power,
infrastructure, or cost of culture diagnostic materials and other issues (12).Hence, Gram stain
is a method of choice for the diagnosis of microorganisms in most health care facilities, as
microbial culture is impossible or not accessible in most of these facilities.

Competency defines the ability to carry out the total performance responsibilities of the given
practitioner’s generic position or competency as the combined knowledge and skill factors
necessary to fulfil work obligations adequately. In other words, competency is the ability to
carry out a specific task within given parameters of control. The competency required for
clinical laboratory personnel reflects performance in many dimensions such as knowledge,
intelligence, technical skills, problem solving abilities, interpersonal skills, and skills in oral
and written expression (13, 14, 15 ).

2
1.2 Statement of the problem

It has been reported that preventable medical errors are responsible for the death of 400,000
Americans annually, the third highest cause of mortality after heart disease and cancer (16). If
60 to 70% of medical decisions are based on laboratory results, it stands to reason that
erroneous laboratory results play some role in avoidable patient mortality (17).

The study conducted at USA on four site on gram stain assessment error rate indicates, For
sites B and C, the majority of discrepant results (79% and 85%, respectively) were Gram
stain negative/culture positive, while for sites A and D, discrepant results were primarily
Gram stain positive/culture negative (85% and 61%, respectively) So, Clinical
microbiologists face similar obstacles in attempts to characterize the incidence and nature of
errors in their laboratories. Many of the tests performed, such as the Gram stain, are prone to
variability and differences in interpretation between technologists (11).

In Ethiopia, even though gram stain is the method of choice for diagnosis of microorganisms
to guide initial choice of antibiotic therapy as stated above, there was a gap between higher
institutions to produce medical laboratory professionals with usable knowledge and practical
skills(18), lack of health information resources(19), no evidence of refresher training and
supervision on gram stain examination and interpretation, low level but progressive
Strengthening Laboratory Management Towards Accreditation (SLMTA) practice(20,21) and
lack of continuing professional development(CPD) program. Hence, due to these gaps Gram
stain examination and interpretation by medical laboratory professionals is low quality and
inconsistent. Lack of proper gram stains examination and interpretation could lead clinicians
to miss diagnose and miss treat patients, which could cause drug resistance, resource-
wastage, and suffering and possibly death of patients and others (9, 22).

In Ethiopia, a study conducted on 12 hospital laboratories participated on the national PT

program from 6 cycles of 2012 and 2013 G.C., Gram stain had high PT failure rates from 20
test parameters. In this study Gram stain failure rate were 88.9% (23).So, this study indicates
there is high knowledge and skill gap on gram stain interpretation in Ethiopia including study
area in particular.

3
In Africa including in Ethiopia there were also limited accessible studies in the scientific
literature that reported competency of medical laboratory professionals on gram stain
examination and interpretation. This indicates that the area was not enough examined by both
researchers and responsible bodies for continuing professional development and quality
medical laboratory services. Therefore, there was lack of scientific evidence on competency
of medical laboratory professionals on Gram stain examination and interpretation in Ethiopia
and in the study area in particular.

4
1.3 Significance of the study

Competency assessment it is a part of problem analysis and becomes a key tool in identifying
errors through quality assurance process and also preventing from recurring. Competency
assessment procedures can help to identify problemsoccurring in the technical aspects of
laboratory practice and assess performance deficiencies before they develop into major
problems. Competency assessment is also an opportunity to provide continuing education and
performance feedback to employees and to document valuable objective information for
performance evaluations. It should and can be used as a positive experience that helps to
ensure that employees and employers can perform assigned tasks.The result of this study will
contribute in provision of concrete data about the Gram stain competency assessment and
associated factorson clinical laboratories in the study area.It will also enable to design
strategies for enhancing patient safety and reduce laboratory error because of significant
laboratory errors have an impact on patient outcome, in some cases leading to erroneous
medical interventions. In addition to this result will be useful for employee to evaluate own
strength and weakness to perform required tasks, and also encourages employees to read and
review carefully of laboratory policies and procedures.

5
2. Literature review
A cross sectional study conducted at USA in 2014 by Goodyear N.on gram stain competency
assessment, out of 278 laboratory staff from 40 laboratories that are participate on the study,
from 40 multiple questions for assessment of knowledge the mean score is 88%(range 63% -
98%), skill level of laboratory professional, mean of identification of host cells is 93%, other
cells like yeast cells, 92%, gram positive 90 % and gram negative 88%. Mean of categorized
by type of interpretation, a laboratory professional who identify by structure like bacterial
morphology feature 91% and identify by Name genus and species 87% (22).

In Ontario, Canada, a study conducted on competency assessment practices on 112 licensed

ontario microbiology laboratories those are participated in EQA (proficiency testing). Out Of
the 111 responding laboratories, 6(5.4%) indicated they did not have a formal evaluation
program since they perform only limited bacteriology testing. Of the remaining 105
respondents, 87% perform evaluations at least annually or every 2 years, and 61% include
any test or task performed, whereas 16% and 10% focus only on problem areas and high-
volume complex tasks, respectively. A survey respondents indicated that the most common
competency issue requiring remediation was associated with Gram staining and interpretation
(30).

The cross sectional studyconducted by Samuel et al, at USA in 2016 focused on several
factors that primarily contributeto errors in the process of Gram stain, including poor
specimen quality, smear preparation, and interpretation of the smears. The number of
specimens during the evaluation period ranged from 976 to 1,864 specimens per site, and
there were a total of 6,115 specimens. Gram stain results were discrepant from culture for 5%
of all specimens. Fifty-eight percent of discrepant results were specimens with no organisms
reported on Gram stain but significant growth on culture, while 42% of discrepant results had
reported organisms on Gram stain that were not recovered in culture. Upon review of
available slides, 24% (63/263) of discrepant results were due to reader error, which varied
significantly based on site (9% to 45%) (24).

In the U.S., a cross sectional study conducted in 1996, a study surveyed a total of 552
institutions and, showed that 89.2% of institutions had a written competency plan and of
those, 90.3% used their plan for microbiology. The study reported that approximately 98% of
institutions review employee competency at least annually. Of all laboratories surveyed,

6
87.5% perform direct supervision, 77.4%review test or QC results, 60% review instrument
preventive maintenance, 52.2% conduct written testing, and 20.8% perform other methods of
assessment as a competency tool. For this study, measuring of adherence to the laboratory’s
own competency plan found that 89.7% assessed using direct observation, 85.8% assessed by
reviewing QC and patient test results, 78% assessed by reviewing instrument records, and
74% assessed using written testing; 90.4% of new employees were assessed as indicated per
policy, and 90% of employees were found to have responded satisfactorily to a written
competency assessment regarding specimen processing. In these institutions failure to
comply with the laboratory’s own competency plan ranged from 1 to 6.4%, and employees
who failed competency assessment were not allowed to continue their usual work in 8.6% of
institutions (25).

An assessment of medical laboratory professionals was done in the U.S. by using sets of
direct Gram stained smears for which the culture results were known. For this study accuracy
of identification of Pseudomonas aeruginosa was 60% for the first survey conducted in 1987
but improved progressively to over 80% in 1988 and 1989 until a decline was observed in
1990. According to this study, this was correlated with an increase in the number of untrained
personnel and indicated a greater need for in-service education in interpretation of gram-
stained smears (26).

Another study was conducted in the U.S., by reviewing major errors in Gram stain reports
from positive blood cultures during a 23-month period. The study reported that blood cultures
were misread for 57 (0.7%) of 8,253 patients. Of 5,885 read as gram-positive cocci, 6 (0.1%)
had only gram-negative organisms by culture, 3 of which were Acinetobacter species. Of
1,959 read as gram-negative bacilli, 25 (1.3%) had only gram positive organisms by culture.
Of these, 9 were Bacillus and 2 were Clostridium species. Non recognition of mixed Gram
stains accounted for 28 errors that most often were associated with a reading of gram-positive
cocci (9).

In other study in the U.S., gram stain results were discrepant from culture for 5% of all
specimens. Fifty-eight percent of discrepant results were specimens with no organisms
reported on Gram stain but significant growth on culture while 42% of discrepant results had
reported organisms on Gram stain that were not recovered in culture. Upon review of
available slides, 24% (63/263) of discrepant results were due to reader error, which varied

7
significantly based on site (9%-45%). The Gram stain error rate also varied among sites,
ranging from 0.4% to 2.7% (11).

Cross sectional study conducted in Ethiopia in 2017 they used both knowledge and skill tests
to assess competence of medical laboratory professionals on gram stain examination and
interpretation. Out of 190 study participants that are administered 15 theory knowledge test
questions, the minimum and maximum score results were 3(20%) and 13(86.7%) respectively
and also 55(28.9%), 131(68.9%) and 4(2.1%) participants scored with low, medium and high
knowledge level respectively. Fromall 1140 observations for skill of medical laboratory
professionals on gram stain examination and interpretation, they found, 44 observations (4%)
with major errors and 321 observations (28%) with very major errors. Of all observations,
321(28.2%) reported without grading, 39 (3.4%) reported gram positive bacteria as gram
negative bacteria and 15(1.4%) reported gram negative bacteria as gram positive bacteria.
One hundred forty eight observations (12.9%) also reported without grading with gram
positive bacteria reported as gram negative bacteria while 43 observations (3.7%) reported
without grading with gram negative bacteria reported as gram positive bacteria (27).

Therefore this study will provide valuable information on gram stain competency on
laboratory professional in mekelle city health facility.

In Ethiopia, there are limited literatures related to our study. Therefore this study will provide
baseline information on gram stain competency of laboratory professional in Mekelle city
health facilities.

8
3. Objective

3.1. General objective:

To assess gram stain competency and associated factor among laboratory professional in
clinical laboratories at Mekelle city health facility

3.2. Specific objectives:

 To assess the knowledge of Medical Laboratory Professionals on gram stain

examination at Mekelle city health facility
 To assess the skill of Medical Laboratory Professionals on gram stain examination
at Mekelle city health facility

 To identify factors associated with competency of medical laboratory

professionals on gram stain at Mekelle city health facilities

9
4. Materials and methods
4.1. Study Area

The study was conducted at Mekelle city, Tigray, north Ethiopia. Mekelle formerly the
capital of Enderta awraja in Tigray, is currently the capital city of Tigray National Regional
State. It is located around 780 kilometres north of the Ethiopian capital Addis Ababa, with an
elevation of 2,254 meters (7,395 ft) above sea level. Administratively, Mekelle is considered
a Special Zone, which is divided into seven sub-cities. Mekelle is the economic, cultural, and
political hub of northern Ethiopia. Based on the 2007 Census conducted by the Central
Statistical Agency of Ethiopia (CSA), this town has a total population of 215,914 people
(104,925 men and 110,989 women).According to Tigray regional health biro, This city has
77 health facility, fromthose 62 are private health facility(4 primary hospitals, 4 lower,20
medium, 34 higher/specialty clinic), one uniformed hospital and 14 are governmental health
facility those are 9 health center (Quiha health center, Aynalem health center, Kasech health
center, Mekelle health center, Adishendehun health center, Adi ha health center, Semen
health center, Serawat and Lachi health center ), Two primary hospital (HEWO and Adihaki),
Two general hospital (Mekelle and Quiha) and one Federal hospital (Ayder comprehensive
specialized hospital). All health facilities that are present at this city were included in the
study.There was lack of scientific evidence on competency of medical laboratory
professionals on Gram stain examination and interpretation in Ethiopia and in the study area
in particular.

4.2. Study Design and Period

A cross-sectional study design was conducted from January 2020 to April 2020.

4.3 Population
4.3.1 Source population

All Medical Laboratory Professionals who were working in Medical Laboratories of health
facilities at Mekelle city, Ethiopia.

10
4.3.2 Study population

All Medical Laboratory Professionals who were working Medical Laboratories of Health
facility in Mekelle city and fulfilled the eligibilitycriteria.

4.4. Eligibility criteria

All laboratory professionals working permanently in Mekelle city health facility during study
period was eligible for the study.

4.5. Study variables

4.5.1. Dependent variables:

Competency level of Medical Laboratory Professionals

4.5.2. Independent variables:

 Sex

Age

Qualification

Experience

Previous training

Health institution type

Higher Institutions(private,governmental)

competency assessment(supervision)

Health information Resource

 Type of Microscope

11
4.6 Sample Size Determination and Sampling Techniques
4.6.1 Sample Size Determination

The totalnumber of medical laboratory professional who was working at governmental and
private health facility wasaround 140 and 62 respectively. This makes a total of 202
professionals.Of those 17, 16, 7 and 7 laboratory, this makes 47 professional was excluded
due to those are on education, break, training and also part time workerduring the study
period respectively, so total number of medical laboratory professional that are working
permanently at government and private health facility during the study period was155, those
are included in the study to increase the reliability and accuracy of the study result.
Preliminary assessment was done to know about the number of medical laboratory
professionals in each health facility before data collection. After preliminary assessment there
were 155 laboratory professional present at the whole health facility that are present at
Mekelle city. Among those 95.5% (148) of themfully participated. The remaining 4.5% were
not willingto participate and lack of time was the most frequent reason given not to
participate.Of those 80, 63 and 5 medical laboratory professional who are from private,
governmental and uniformed health facility respectively.

4.6.2 Sampling Techniques

All laboratory professional who are working permanently at Mekelle health facility was
included using Convenience Sampling technique.

12
4.7. Measurement and Data collection
4.7.1Data collection tools and procedure

Data on Socio demographic characteristics and 25 knowledge assessment question of study

subject were collected byself-administered structured questioner. Thequestionnaire
wasdeveloped based on reviewing different literatures.7 panel slides were prepared to assess
skill of laboratory professionals.Stainedand graded slide were provided to each participant
and then each participant was graded when interpreting one stained slide within five minutes.
Participants were provided 25 structured knowledge questions with background questionnaire
to fill their response within 30 minutes and also monitored the participant while filling the
questionnaire so as to not use reading material and discuss with their friends.Functionality of
microscope was checked by senior laboratory professional by using control slide that are
prepared from ATCC.The data from gram stained slide identification and 25 structured
knowledge questions with background questionnaire from each study participant were
collected by the researcher. Generally, Gram stains reporting criteria per oil immersion:

Grade Description Cells Bacteria

1+ Rare <1 <1

2+ Few 1–5 2–10

3+ Moderate 6–10 11–50

4+ Many >10 >50

Based on the reading of more than 10 fields (29).

13
4.7.2 Panel Slide Preparation and Distribution

Senior microbiologists that are present at Ayder comprehensive specialized hospital and the
principal investigator prepared and validated the gram stained panel slides.Patient sample for
no microorganisms with cell and samples from known bacterial strains known as American
Type Culture Collection (ATCC) were used for preparation of gram stained panel slides.The
known bacterial strain was sub cultured on blood agar. From growth on blood agar, gram
stained panel slides was prepared. All slides were stained by gram staining procedure. Senior
microbiologist interpret the prepared gram stain smears using investigative criteria for the
presence or absence of gram stain findings (bacteria, yeasts and cells) and also quantification
of findings (few, moderate and many). Senior microbiologist were validated the gram stain
interpretation agreement with the culture growth on blood agar. For this study, seven gram
stained panel slide types were used and the composition of test panels prepared in
Microbiology Laboratory Department and those panel slides have Gram positive cocci in
cluster (S.aureus), Gram positive cocci in chain(S.pyogenes), Gram
negative(N.gonorrhoeae)and Gram positive(S.pnemonia) diplococcic and Gram positive
(bacillus) and gram negative (E.coli) rods from ACSH, and no microorganisms with many
pus cells from patient sample. Panel of slides were graded as few, moderate and many with
different interpretation. Next to preparation, the slides and reporting format was arranged,
packed and distributed to the participant by the researcher.

4.8 Data management and Data Quality Assurance:

The following precautions were considered so as to assure the quality of the data. The data
were collected by the researcher. By using standard data collection material, preparing of
quality control from ATCC for checking of Microscope functionality before assessing the
performance of laboratory personnel and pre testing of the questioner’s data at wukro general
hospital, checking the completeness of questionnaires. Based on the findings of the pre-test,
some questions were modified. Furthermore data were checked during entry into the
computer before analysis. After preparation of panel slide from ATCC, all slides were stained
by gram staining procedureThen Senior microbiologist were validated the gram stain
interpretation agreement with the culture growth on blood agar and alsointerpreted the
prepared gram stain smears using investigative criteria for the presence or absence of gram
stain findings (bacteria, yeasts and cells) and also quantification of findings (few, moderate
and many).

14
4.9Data analysis and presentation:
Data obtained was checked for completeness and Qualitative data from the questionnaires
coded and entered into SPSS version 23 data analysis. Descriptive statistics were used to
summarize socio demographic characteristics of study participant.The univariate analysis
such as percentage and frequency distribution of different characteristics of the questionnaire
was analyzed. Bivariate analysis was used to see the association of independent with the
dependent variable but there were no difference with multivariate analysis.A chi square
model was employed to determine associations with dependent and independent variables.
Percent of correct response to a set of 25 knowledge questions were graded as follows: 59 %
or below (14.7/25) as low, 60-80 % (15-20/25) as medium and above 80 % (>20/25): as high
knowledge level. Similarly, percent of correct responses to a set of 7 skill related questions
were graded as follows: those who perform correctly in 59% or below (4.13/7) as low, 60-80
% (4.2-5.6/7) as medium and above 80 % (>5.6/7) as high skill level(28). Major error, minor
error, very major error, maximum score, minimum score were analyzed for skill tests and
also participants grouped in to low, medium and high skill level. For 25 knowledge questions
the analyses were performed for maximum score and minimum score of questioner answers
and also classify participants to low, medium and high knowledge level. Valueof <0.05 was
considered statistical significant. Participants’ knowledge about gram stain was assessed
using 25questions. Define gram stain, difference between cell structure of gram positive and
gram negative bacteria, important of decolourization in gram staining, Why is the gram stain
considered a differential stain and important of knowing gram positive or gram negative
bacteria. Those questions had a coded in spss (minimum correct response coded by ‘yes’ and
wrong or don’t know response had a code of ‘No’). The rest had single answers, so giving a
value as a choice question.

15
4.10 Operational Definitions:

Competency:is the ability of personnel to apply their skill, knowledge, and experience to
perform their laboratory duties correctly.

Uniformed Hospitals: Armed Force Referral and Teaching Hospital and Police Hospital

High knowledge and Skill:when Percent of correct response> 80 %. (28)

Medium knowledge and Skill:when the percent of correct response 60 – 80 %.( 28)

Low knowledge and Skill: when the percent of correct response ≤ 59 %. (28)

Gram stain interpretation minor error: report the presence of polymorph when not present
(29).

Gram stain interpretation major error: report the presence of an organism not present or
failure to report the presence of 1+ to 2+ polymorphs (29).

Gram stain interpretation very major error: failure to report the presence of an organism
and failure to report the presence of 3+ to 4+ polymorphs (29).

16
4.11 Ethical considerations

This study was ethically approved by the “departmental research and ethical review
committee” (DRERC) of the department of medical laboratory science. Permission letter was
written fromTigray regional health biro. All the collected data was kept confidentially by
using codes instead of any personal identifiers. It is also cleared that participation fully based
on the willingness of participants using written consent.

4.12 Dissemination of the result

The result of this study will be disseminated or communicated to Addis AbebaUniversity

College of Health Science, department of medical laboratory science and also governmental
and private health institution and other concerned bodies that are present atmekelle city
through reports and publication on an appropriate journal or by presentations of the findings
in appropriate scientific conferences.

17
5. Result

5.1 Socio-demographic characteristics of the studyparticipant

During study time, there were 77 health facilities; of them 62 were private, 14 governmental
and also one uniformed health facility. Among 62 health facilities, 58 of them was clinics, the
rest one was primary hospitals and also among 14 governmental health facility, 9 was health
center, one specialized hospital, two primary hospitals and two referral hospitals.

Of 155 medical laboratory professionals, 95.5% (148) of them fully participated. The
remaining 4.5% were not willingto participate and lack of time was the most frequent reason
given not to participate.Out Of148 respondents 78(52.7%) were males. regarding education,
67(45.3%),73(49.3) and 8(5.4) were degree, diploma and masters holder respectively.Fifty-
nine (39.9%)of the respondents were in the age group of 26-30 years.The present study
indicates Most of the study participants 107(72.3%) had work experienceof ≤ 9 years.Nearly
half, 81(54.7%) participants were from private health facility andseventy-two (46.2%)
respondents were who graduate from Government University.Most 109(73.6%) of respondent
did not participate in EQA and among study participants most of them 106(71.6%) did not
get regular competency assessment (supervision). Of respondent 142 (95.9%) were not get
training related to gram stain. During the study time, 109(73.6%) respondents were from
health facilities that are not participating on accreditation program.(Table 1)

18
 Table 1:Socio demographic characteristics of medical Laboratory professionals in
Mekelle city health facility,Tigray, Ethiopia 2020(N=148)
Variable characteristics Number percent
Male 78 52.7
Sex
Female 70 47.3
Age in years ≤ 25 27 18.2
26-30 59 39.9
31-35 36 24.3
≥36 26 17.6
Experience in years 0-9 107 72.3
10-19 35 23.6
≥20 6 4.1
Educational level Diploma 73 49.3
Degree 67 45.3
Masters 8 5.4
Health facility type Government 62 41.9
private 81 54.7
uniformed 5 3.4
EQA Yes 39 26.4
No 109 73.6
Not participated 109 73.6
Current SLIPTA level
Star 0 4 2.7
Star 1 5 3.4
Star 2 11 7.4
Star 4 19 12.8
Training related to gram stain Yes 6 4.1
No 142 95.9
Competency assessment(supervision) Yes 42 28.4
No 106 71.6
Licence provider institution type Government college 59 39.9
Government university 72 48.6
Private college 17 11.5
Over all total 148 100

19
 5.2 Resource access of study participants
Out of all study participants, there was 102(68.9%) laboratory professional who has health
information resource in their laboratory section. Among all respondents, 53(35.8%) of study
participants used Olympus microscope. (Table 2)

Table 2: Resource access of study participants at Mekelle city health facility, Tigray,
Ethiopia 2020
Variable characteristics Number percent
Health information resource Yes 102 68.9
No 46 31.1
Total 148 100
Types of microscope Olympus 53 35.8
Novel 40 27.0
primo star 21 14.2
Gemmy and heuer 19 12.8
Other 15 10.1
Total 148 100

20
5.3Knowledge of medical laboratory professional

For the 25 theoretical knowledge questions administered to 148 participants, the minimum
score was 0(0%) and the maximum score was 24(96%).A number of medical laboratory
professionals who score 0 of 25 knowledge assessment questionswere 7(4.7%). Among
participants, the knowledge score was low for 95 (64.2%), medium for 49(33.1 %) and high
for 4 (2.7 %).From study respondents with low knowledge levels, most of the respondents
59(62.1%) with low knowledge levels were those who had diploma. Analyses were carried
out to examine the association between different factors and knowledge of study participants
on gram stain examination. Education level, SLIPTA level of health facility, Licence
provider institution type and sex had statistically significant association with knowledge level
of study participants (p value <0.005).Out of148study participant four respondents had high
knowledge level, among those two of them had degree holder and one haddiploma and
masters holder.(Table 3)

Out of four study participants who had high knowledge level, three of them were from health
facility that had star 4 level of SLIPTA, the rest one was from a health facilities that are not
participating on accreditation. About 79(83.2%) of respondentswho had low knowledge level
was from health facility that are not participating on accreditation.Of respondents with high
knowledge level (4), 3(75%) of them were respondents who graduate from government
university and also all respondents who graduates in private college had low knowledge level.
As the present study indicates among low and high knowledge level participants, 55(57.9%)
and 3(75%) were Female and Male respectively.(Table 3)

Age, microscope type, health facility type, health information resources, EQA
participation,training, competency assessment and experience had no significant association
with knowledge level of medical laboratory professionals on Gram stain examination.(Table
3)

21
 Table 3: Knowledge of medical laboratory professional related to background
characteristics in mekelle city health facility, Tigray, Ethiopia, 2020 (n=148)

Knowledge level
Variable characteristics Low Medium High
No(%) No (%) No (%) Total X2 P-value
Age <25 26(17.6%) 1(0.7%) 0(0%) 27(18.2%) 5.359 0.499
26-30 47(31.8%) 11(7.4%) 1(0.7%) 59(39.9%)
31-35 31(20.9%) 4(2.7%) 1(0.7) 36(24.3%)
≥36 23(15.5%) 3(2%) 0(0%) 26(17.6%)
Sex Male 40(27%) 35(23.6%) 3(2%) 78(52.7%) 11.971 0.003
Female 55(37.2%) 14(9.5%) 1(0.7%) 70(47.3%)
Health Government 33(53.2%) 27(43.5%) 3(2%) 62(41.9%) 5.827 0.212
facility type Private 58(71.6) 21(25.9) 1(1.2%) 80(54.1%)
Uniformed 4(80%) 1(20%) 0 5(3.4)
Licence Government college 43(29.1%) 15(10.1%) 1(0.7%) 59(39.9%) 19.078 0.001
provider Government university 35(23.6%) 34(22.9%) 3(2%) 72(48.6%)
institution Private college 17(11.5%) 0(0%) 0(0%) 17(11.5%)
type
Experience 0-9 73(49.3%) 32(21.6%) 2(1.4%) 107(72.3%) 3.957 0.412
in years 10-19 19(12.8%) 14(9.5%) 2(1.4%) 35(23.6%)
≥20 3(2%) 3(2%) 0(0%) 6(4.1%)
Educational Diploma 59(39.9%) 13(8.8%) 1(0.7%) 73(49.3%) 27.169 0.000
level Degree 36(24.3%) 29(19.6%) 2(1.4%) 67(45.3%)
Masters 0(0%) 7(4.7%) 1(0.7%) 8(5.4%)
EQA Yes 22(14.9%) 16(10.8%) 1(0.7%) 39(26.4%) 1.506 0.471
participation No 73(49.3%) 33(22.3%) 3(2%) 109(73.6%)
Not participated 79(53.4) 28(18.9%) 1(0.7%) 108 22.381 0.004
Current Star 0 2(1.4%) 2(1.4%) 0 4(2.7%)
SLIPTAlevel Star 1 4(2.7%) 1(0.7%) 0 5(3.4%)
Star 2 6(4.1%) 5(3.4%) 0 11(7.4%)
Star 4 4(2.7%) 13(8.8%) 3(2%) 20(13.5%)
Training Yes 2(1.4%) 3(2%) 1(0.7%) 6(4.1%) 5.978 0.050
No 93(62.8%) 46(31.1%) 3(2%) 142(95.9%)
Competency Yes 28(18.9%) 13(8.8%) 1(0.7%) 42(28.4%) 0.161 0.923
assessment No 67(45.3%) 36(24.3%) 3(2%) 106(71.6%)
Health Yes 59(39.9%) 39(26.4%) 4(2.7%) 102(68.9%) 6.469 0.039
information No 36(24.3%) 10(6.8%) 0 46(31.1%)
resource
Olympus 33(22.3%) 18(12.2%) 2(1.4%) 53(35.8%) 11.624 0.071
Types of Novel 26(17.6%) 13(8.8%) 1(0.7%) 40(27%)
microscope Primo star 8(5.4%) 12(8.1%) 1(0.7%) 21(14.2%)
Gemmy and heuer 15(10.1%) 4(2.7%) 0(0%) 19(12.8%)
Other 13(8.8%) 2(1.4%) 0(0%) 15(10.1%)
Over all total 95(64.2%) 49(33.1%) 4(2.7%) 148(100%)

22
 5.4Response of medical laboratory professional on each theoretical
knowledge question about Gram stain

Out of all participants, 110(74.3%) respondents score correct answer on question of what is a
primary stain in gram stain technique, and also 124(83.8%) respondents score incorrect
answer on the question of what would happen if iodine were missed during gram staining
procedure.(Table 4)

Table 4: Participants’ knowledge about Gram stain among medical laboratory

professionals at Mekelle city, Tigray, Ethiopia, 2020 (n= 148)

No Item Yes(Corr No(Incorre

ect ) ct )
1 Define gram stain 67(45.3%) 81(54.7%)
2 One gram positive bacteria 79(53.4%) 69(46.6%)
3 The difference between cell structure of gram positive and gram negative bacteria 28(18.9%) 120(81.1%)
4 Important of decolourization in gram staining 39(26.4%) 109(73.6%)
5 Possible cause of false gram reaction results 108(73%) 30(27%)
6 Appearance of Gram positive bacteria 41(27.7%) 107(72.3%)
7 Appearance of Gram negative bacteria 88(59.5%) 60(40.5%)
8 bacteria’sdecolorized by Acetone alcohol & contain a counter stain colour 90(60.8%) 58(39.2%)
9 Bacteria resist decolourization& retain the colour of the primary stain 90(60.8%) 58(39.2%)
10 A counter stain in Gram stain technique 106(71.6%) 42(28.4%)
11 A primary stain in Gram stain technique 110(74.3%) 38(25.7%)
12 one possible morphologies of bacteria 77(52%) 71(48%)
13 Consequence of if iodine were missed during gram staining 24(16.2%) 124(83.8%)
14 The order of reagents used in the Gram stain 82(55.4%) 66(44.6%)
15 Use of heat fixative after smearing the sample before we use reagent to stain bacteria 80(54.1%) 68(45.9%)
16 Typical morphology of staphylococcus aures 51(34.5%) 97(65.5%)
17 The gram stain considered a differential stain 12(8.1%) 136(91.9%)
18 Gram positive cocci in pairs mean(suggestion/interpretation) 57(38.5%) 91(61.5%)
19 Gram positive cocci in cluster mean(suggestion/interpretation) 51(34.5%) 97(65.5%)
20 Important of knowing gram positive or gram negative bacteria 68(45.9%) 80(54.1%)
21 A counter stain other than safranin be used in gram staining 21(14.2%) 127(85.8%)
22 A primary stain other than crystal violet be used in gram staining 20(13.5%) 128(86.5%)
23 Consequence of if you reverse crystal violet and safranin stains 28(18.9%) 120(81.1%)
24 Safranin basic or Acidic dye 77(52%) 71(48%)
25 One gram negative bacteria 59(39.9%) 41(60.1%)

23
5.5The level of the skills of the medical laboratory professional on gram
stain examination
Among all respondents assessed for skills in gram stain examination, one hundred twenty-
seven 127(85.2%), Nineteen 19(12.8%) and 2(1.3%)participants scored low, medium and
high skill level respectively.The level of skills of medical laboratory professionals
variedaccording to educational level, SLIPTA levelandtraining (P < 0.05). (Table 5)

During study time, regarding to educational levelamong respondents with low skill levels,
most of the respondents were diploma and degree level71(55.9%) and 52(40.9%)
respectively.Of respondents who had low skill levels, 101(79.5%)was from health facility
that are not participating on accreditations. Inaddition to theseparticipants from SLIPTA level
of stare 0 and 1 health facility had no high skill level but respondents with high skill level
were those are from SLIPTA level 2 and 4 health facility. (Table 5)

Out of high skill level participants, all 2(100%) had a gram stain training certificate whereas
of all participants with low knowledge level,almost all of them 124(97.6%) had no training
on gram stain. Among all study participants, 83.8% had no training certificate on gram stain
and had low knowledge level.Of respondents with low knowledge levels were a respondents
who get one 2(33.3%) and two 1(16.7%) gram stain training certificates.(Table 6)

Table 5: Skill of medical laboratory professional on each slides about Gram stain

Among all respondents, 120(81.1%) participants incorrectly report gram stain slide 6 (gram
positive cocci) as gram negative bacteria and also 87(58.8%) participants correctly report
gram stain slide 5 (gram negative rod). (table 5)
Correct In correct
Item
Gss 1 66(44.6%) 82(55.4%)
Gss 2 49(33.1%) 99(66.9%)
Gss 3 70(47.3%) 78(52.7%)
Gss 4 52(35.1%) 96(64.5%)
Gss 5 87(58.8%) 61(41.2%)
Gss 6 28(18.9%) 120(81.1%)
Gss 7 64(43.2%) 84(56.8%)

24
 Table 6: The level of the skills of the medical laboratory professional on gram stain
examination in Mekelle city health facility,Tigray, Ethiopia, 2020

Skill level
Variable characteristics
Low No Medium High
(%) No (%) No (%) Total X2 p-
value
Age <25 26(17.6%) 1(0.7%) 0(0%) 27(18.2%) 5.359 0.499
26-30 47(31.8%) 11(7.4%) 1(0.7%) 59(39.9%)
31-35 31(20.9%) 4(2.7%) 1(0.7%) 36(24.3%)
≥36 23(15.5%) 3(2%) 0(0%) 26(17.6%)
Sex Male 62(41.9%) 14(9.5%) 2(1.4%) 78(52.7%) 5.919 0.052
Female 65(43.9%) 5(3.4%) 0(0%) 70(47.3%)
Health facility Government 50(79.4%) 11(17.5%) 2(3.2%) 63(42.6%) 4.438 0.232
type Private 73(91.2%) 7(8.8%) 0(0%) 80(54.1%)
Uniformed 4(80%) 1(20%) 0(0%) 5(3.4%)
Licence provider Government college 56(37.8%) 3(2%) 0(0%) 59(39.9%) 13.945 0.007
institution type Government university 54(36.5%) 16(10.8%) 2(1.4%) 72(48.6%)
Private college 17(11.5%) 0(0%) 0(0%) 17(11.5%)
Experience in 0-9 92(62.2%) 14(9.5%) 1(0.7%) 107(72.3%) 0.938 0.919
years 10-19 30(20.3%) 4(3.1%) 1(0.7%) 35(23.6%)
≥20 5(3.4%) 1(0.7%) 0(0%) 6(4.1%)
Educational level Diploma 71(55.9%) 2(10.5%) 0(0%) 73(49.3%) 21.907 0.000
Degree 52(40.9%) 13(68.4%) 2(2.9%) 67(45.3%)
Masters 4(3.1%) 4(3.1%) 0(0%) 8(5.4%)
EQA participation Yes 31(20.9%) 7(4.7%) 1(0.7%) 39(26.4%) 1.901 0.387
No 96(64.9%) 12(8.1%) 1(0.7%) 109(73.6%)
Not participated 101(79.5%) 8(5.4%) 0(0%) 109(73.6%) 28.191 0.000
Current SLIPTA Star 0 4(3.1%) 0(0%) 0(0%) 4(3.1%)
level Star 1 4(3.1%) 1(0.7%) 0(0%) 5(3.4%)
Star 2 8(5.4%) 2(1.4%) 1(0.7%) 11(7.4%)
Star 4 10(6.8%) 8(5.4%) 1(0.7%) 19(12.8%)
Training Yes 3(2%) 1(0.7%) 2(1.4%) 6(4.1%) 48.339 0.000
No 124(83.8%) 18(94.7%) 0(0%) 142(95.9%)
If yes, How many One 2(1.4%%) 0(0%) 1(0.7%) 3(2%)
times/certificate Two 1(0.7%) 0(0%) 1(0.7%) 2(1.4%)
Four 0(0%) 1(0.7%) 0(0%) 1(0.7%)
Total 3(2%) 1(0.7%) 2(1.4%) 6(4.1%)
Competency Yes 36(24.3%) 5(3.4%) 1(0.7%) 42(28.4%) 0.500 0.779
assessment(super No 91(61.5%) 14(9.5%) 1(0.7%) 106(71.6%)
vision)
Health Yes 83(56.1%) 17(11.5%) 2(1.4%) 102(68.9%) 5.403 0.067
information No 44(29.7%) 2(1.4%) 0(0%) 46(31.1%)
resource Total 127(85.8%) 19(12.8%) 2(1.4%) 148(100%)
Types of Olympus 43(29.1) 8(5.4%) 2(1.4%) 53(35.8%) 7.763 0.256
microscope Novel 37(25%) 3(2%) 0(0%) 40(27%)
Primo star 16(10.8%) 5(3.4%) 0(0%) 21(14.2%)
Gemmy and heuer 18(12.2%) 1(0.7%) 0(0%) 19(12.8%)
Other 13(8.8%) 2(1.4%) 0(0%) 15(10.1%)
Over all total 127(85.8%) 19(12.8%) 2(1.4%) 148(100%)

25
 5.6Errors of Medical Laboratory Professionals on gram stain examination

During the study time, results from a total of ( 148 x 7=1036) observation, 437(42.2%
)observation with major error and 308 (29.7%) observation with very major error. 249
observations (24%) reported gram positive bacteria as gram negative bacteria and 106
observations (10.2 %) reported gram negative bacteria as gram positive bacteria.(Table 7)

About 55.4 % of laboratory professionals have reported negative slides (slide 1) as false
positive (gram positive and gram negative). 49(33.1%) of the participants correctly reported
the bacteria of slide 2. Slide 3 contains Gram positive rod (many), 70(47.3%)of the
participants correctly reported the bacteria. Similarly 52(35.1%) of the participants correctly
reported the bacteria of slide 4. Out of respondents87(58.8%),28(18.9%), and
64(43.2%)correctly reported bacteria of slide 5, 6 and 7 respectively. (Table 7)

Table 7: The Skill level of Medical Laboratory Professionals on gram stain examination
at Mekelle city health facility, Tigray, Ethiopia 2020

Characteristics Error type

Slide Gram No. of No. of Minor Major V.major
number reaction participants participants
correctly correctly Other
Total report report cells(PMNS)
No posneg Morphology Density of
both of bacteria bacteria
GSS 1 66 48 34 0 148 - - 11 - 82 137
GSS 2 25 49 73 1 148 64 54 1 1 73 25
GSS 3 28 70 47 3 148 75 64 0 0 47 28
GSS 4 37 59 52 0 148 38 28 0 0 59 37
GSS 5 14 47 87 0 148 79 19 1 1 47 14
GSS 6 26 28 92 2 148 57 44 1 1 92 26
GSS 7 41 64 37 6 148 35 21 1 1 37 41
Total 237 365 422 12 1036 4 437 308
Notice: Gss 1: no gram rxn with many PMNCs, Gss 2: gram +vecocci (s.aures), Gss 3: gram
+ve rod, Gss 4: gram –ve diplococcic, Gss 5: gram –ve rod, Gss 6: gram
+vecocci(s.pyogens), Gss 7: gram +vediplococci

26
6. Discussions
The goal of competency assessment is to improve the laboratory’s performance by
identifying areas requiring education and/or training of the staff and thus ensuring patient
safety. Ongoing competency assessment outside initial training is not as universally
implemented and, in some cases where it exists, may be limited in scope and intensity.
Performing competency assessment as the need arises is a reactive rather than a proactive
approach. The goal should be to detect problems before they happen. Ongoing assessment
needs to be incorporated into a laboratory’s quality management system. Quality
laboratorytesting is an essential building block of the clinical diagnosis scheme, infectious
disease surveillance, and the development of public health policy. Following good laboratory
practices leads to reliable and accurate test results, which in turn fosters good patient care and
promotes a positive attitude toward testing from providers’ and patients’ perspectives.

In this study, we used both knowledge and skill tests to assess competence of medical
laboratory professionals and associated factors on gram stain examination.This study showed
Among 148 participants, the knowledge score was low for 95 (63.8%), medium for 49(32.9
%)and high for 4 (2.7 %) and also the skill score was low for 127(85.2%)%, medium for
19(12.8%) and high for 2(1.4%) so, this finding revealed that participants with
lowknowledge level were higher than the study conducted at Adis Abeba, regrading to skill
level, as study at Adis Abeba revealed that participants with high skill level was 33.7% , so
this indicate the knowledge and skill level of participants was better than our studies this may
be due to all participants was from hospitals(27) but in both studies,the participants with high
knowledge level were very few. This may be due to lack of regular competency assessment,
training and also lack of attention from the responsible body. When we seethe skill test of our
study,out of all participants 13.4% and 21.4%of respondents correctly identify gram negative
and gram positive respectively this may be due to lack of proper funding,
adequate training for laboratory workers and systematic management of work. A study in the
U.S showed that participants score of gram negative(88%) and gram positive (90%),which
was higher than our study (22) so, this finding was it may be due to adequate training,
supervision,high infrastructure of laboratory facility and also advanced technology.

Our study indicate that from 25 knowledge assessment questions the mean score was 44.3%
(range 0% - 96%), mean score was lower than the finding in USA in 2014 by Goodyear

27
N(22). This may be due to continuous training, concerning body giving an attention on this,
enough funding budget to improve quality and also advanced set up.

Analyses were carried out to examine the association between different factors and
knowledge of study participants on gram stain examination. So our study found Sex, Licence
provider institution type, Education level and SLIPTA level of health facility had statistically
significant association with knowledge level of study participants.Level of knowledge of
participants regarding to educational level was low for diploma holder than degree and
master’s holder. So concerning body should give education opportunity to medical laboratory
professional in order to upgrade themselves. From study respondents with low knowledge
level, most of the respondents 59(62.1%) with low knowledge level were those who had
diploma, this number was higher than the study conducted at Adis Abeba (27). This may be
due to most study participants were diploma level or it may be due to most participants was
from private health facility. Of respondents with high knowledge level (4), all of them were
respondents who graduate from government institution this was similar with the study
conducted at Adis Abeba (27). Both study results indicate may be due to responsible body not
give attention on private higher institution. When we compare government institution
(university and college), 3(75%) of them was from government university this may be due to
responsible body gives attention on government university rather than college. In addition to
this when we compare Government College and private college, all respondents who
graduates in private college had low knowledge and skill level but not participants from
Government College, which had medium and high knowledge level and also medium skill
level.This may be due to lack of attention of responsible body on private higher institution.

This study showed that Out of four study participants who had high knowledge level, three of
them were from health facility that had star 4 level of SLIPTA, the rest one was from a heath
facility that are not participate on accreditation and also of the low level respondents about
79(73.1%) of respondents who had low knowledge level was from health facility that are not
participate on accreditation but only 4(4.2%) respondents who were from health facility that
had star 4 on SLIPTA level so, this indicates participation on accreditation has an impact on
knowledge of medical laboratory professional.

Among 148 respondents assessed for skills in gram stain examination, one hundred twenty
seven 127(85.2%), Nineteen 19(12.8%) and 2(1.3%) participants scored low, medium and
high skill level respectively in addition to this high and low score for skill was 6(85.7%) and

28
0(0%) from all 7 skill tests. The level of skills of medical laboratory professional varied
according to educational level, accreditation status and training.During study time, regarding
to educational level among respondents with low skill level, most of the respondents were
diploma and degree level 71(55.9%) and 52(40.9%) respectively. Participants who were
diploma and masters level had no high skill level. Of respondents who had low skill level,
101(79.5%) and also no high skill level was from a health facility that are not participating on
accreditations in addition to this SLIPTA level of stare 0, 1 had no high skill level. From
study participants with high skill level (2), all respondents 2(100%) were those were from
SLIPTA level star 2 and 4 health facility. Although related literature was not accessed, our
study showed that participation in accreditation and also increase step of SLIPTA level had
an impact on the skill of medical laboratory professionals.

This study showed that among all study participants, 83.8% had no training on gram stain and
had low knowledge level. From all study participants with high skill levels (2), all of them
had gram stain training certificate. Of respondents who get training certificates with low skill
levels were respondents who get one 2(1.4%) and two 1(0.7%) training certificates.
Participants who get four training certificate had no low skill on gram stain examination. The
study done in US by using gram stained smear of known culture result, accuracy of
identification of Pseudomonas aeruginosa was 60% but after 1-2 years of in service training it
improved progressively to over 80% (26). So this study indicates if there is continuous
training it can be improve our skill level. Generally, our study showed that this area had a
lack of attention from the concerning body. Doing competency assessment definitely
improves the quality of the laboratory services provided using gram staining methods.

29
7. Limitation

Too little literature was available And Shortage of recently conducted studies are some of the
limitation.

8. Conclusion
Among participants, the knowledge score was low for 95 (64.2%) and one hundred twenty
seven 127(85.2%) for skill score, So this showed that the majority of medical laboratory
professional had low knowledge and skill in gram stain examinations this may be due to most
participants had no get training, were from heath facility that are not participate on
accreditation, diploma level and also it may be due to the professionals focus on other
laboratory technique.

As the present study indicates, License provider institution type, sex, SLIPTA level of health
facility and educational level affect knowledge level of medical laboratory professional on
gram stain examination. Educational level, SLIPTA level of health facility and trainingwere
affect skill level of medical laboratory professional on gram stain examination.As our study
showed most participant with low skill level were working without training this may affect on
gram stain examination. Even though this study did not show association of microscope type
with skill level, the study conducted at Adis Abeba indicates microscope type correlated with
skill of medical laboratory professionals on gram-stain examination.

30
9. Recommendation
 Training and education is needed to reduce error rate of gram stain examination,
those are not only used to improve knowledge and skill level of medical laboratory
professionals or the quality of test results, but also clinicians trust in the laboratory
so, Concerning body like Tigray regional health biro should give education and
training opportunity to medical laboratory professional in order to upgrade
themselves
 It requires to give attention by Concerning body like Tigray regional laboratory
preparing of training on gram stain.
 The WHO AFRO has established a 5-step accreditation process structured around its
core standards for laboratories, which will allow laboratories to gradually receive
credit for improvement and eventually attain accreditation; So Heath facility should
require participation on accreditation.

31
10. Reference

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2. Martínez-Lorente A, Dewhurst F, Dale B. Total quality management: Origins and

evolution of the term. The TQM Magazine. 1998;10.
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263a. Public Law No. 100-578.
4. Adwan G, Shtayah A, Adwan K, Al-Sheboul S, Othman S. Prevalence and molecular
characterization of P. aeruginosa isolates in the West Bank-Palestine for ESBLs, MBLs and
integrons. J Appl Life Sci Int. 2016;8(2):1-11.
5. Beta-lactamases B, d Inhibitor Resistant T. Extended spectrum beta-lactamases.
6. Wilhelm MJ, Sheffield JB, Sharifian Gh M, Wu Y, Spahr C, Gonella G, et al. Gram’s
stain does not cross the bacterial cytoplasmic membrane. ACS chemical biology.
2015;10(7):1711-7.
7. Nagendra S, Bourbeau P, Brecher S, Dunne M, LaRocco M, Doern G. Sampling
variability in the microbiological evaluation of expectorated sputa and endotracheal aspirates.
Journal of clinical microbiology. 2001;39(6):2344-7.
8. Munson E, Block T, Basile J, Hryciuk JE, Schell RF. Mechanisms to assess Gram
stain interpretation proficiency of technologists at satellite laboratories. Journal of clinical
microbiology. 2007;45(11):3754-8.
9. Rand KH, Tillan M. Errors in interpretation of Gram stains from positive blood
cultures. American journal of clinical pathology. 2006;126(5):686-90.
10. Church DL, Don-Joe C, Unger B. Effects of restructuring on the performance of
microbiology laboratories in Alberta. Archives of pathology & laboratory medicine.
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gram stain error rates. Journal of clinical microbiology. 2016;54(6):1442-7.
12. Dagnaw AM, Tiruneh BZ. Most laboratory tests used in the diagnosis of tuberculosis
in Ethiopia are based on direct microscopy: A systematic review. health-care. 2014;3:4.
13. Woodcock S, Fine G, McClure K, Unger B, Rizzo-Price P. The role of standards and
training in preparing for accreditation. American journal of clinical pathology.
2010;134(3):388-92.

32
14. Parikh RP. Competency assessment for medical laboratory practitioners and existing
rules and regulations. Journal of Health Occupations Education. 2000;14(2):6.
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professional competence: building blocks for theory development. Best practice & research
Clinical obstetrics & gynaecology. 2010;24(6):703-19.
16. James JT. A new, evidence-based estimate of patient harms associated with hospital
care. Journal of patient safety. 2013;9(3):122-8.
17. Forsman RW. Why is the laboratory an afterthought for managed care organizations?
Clinical Chemistry. 1996;42(5):813-6.
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laboratory education strengthening enhances sustainable laboratory workforce in Ethiopia.
Human resources for health. 2013;11(1):56.
19. Andualem M, Kebede G, Kumie A. Information needs and seeking behaviour among
health professionals working at public hospital and health centres in Bahir Dar, Ethiopia.
BMC health services research. 2013;13(1):534.
20. Hiwotu TM, Ayana G, Mulugeta A, Kassa GB, Kebede Y, Fonjungo PF, et al.
Laboratory system strengthening and quality improvement in Ethiopia. African journal of
laboratory medicine. 2014;3(2).
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Strengthening Laboratory Management Towards Accreditation (SLMTA) on laboratory
quality management system in city government of Addis Ababa, Ethiopia. The Pan African
Medical Journal. 2015;20.
22. Goodyear N, Kim S, Reeves M, Astion ML. A 2-year study of Gram stain
competency assessment in 40 clinical laboratories. American journal of clinical pathology.
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23. Gurmessa A, Sisay A. Proficiency Test Feedback Utilization at Government Hospitals
Laboratory, Addis Ababa, Ethiopia. J Med Diagn Meth. 2017;6(258):2.
24. Samuel LP, Balada-Llasat J-M, Harrington A, Cavagnolo R. Correction for Samuel et
al., Multicenter Assessment of Gram Stain Error Rates. Journal of clinical microbiology.
2016;54(9):2405.
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Clinical microbiology reviews. 2004;17(3):681-94.

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26. Bartlett RC, Mazens-Sullivan M, Tetreault JZ, Lobel S, Nivard J. Evolving
approaches to management of quality in clinical microbiology. Clinical microbiology
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27. Adugna T. competency assessment on gram stain examination and interpretation among
laboratory professionals(unpublished master’s thesis) Addis Abeba University, Ethiopia:
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health workers in leprosy control activities at public health facilities in Amhara and Oromia
States, Ethiopia. BMC health services research. 2016;16(1):122.

29.Church D, Melnyk E, Unger B. Quantitative Gram stain interpretation criteria used by

microbiology laboratories in Alberta, Canada. Journal of clinical microbiology.
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technologists in Ontario, Canada. Journal of clinical microbiology. 2014;52(8):2940-5.

34
11. Annexes
Annex I: Information Sheet (English Version)
Title of the project:Assessment of Gram stain competency and associated factors in clinical
laboratories in Mekelle City health institute, Ethiopia
Name of Principal Investigator: Fikadu Miruts
Organization: Addis Ababa University
Name of sponsor: Mekelle University
This information sheet was prepared for Medical laboratory professionals who will be
involved in project entitled above. I was tell them about the whole processes that have been
undertaken in the study and requesting them to participate voluntarily.
Description and Purpose of the study
The Gram stain is a classic biological protocol that is still actively used to differentiate
bacteria into two possible classifications: Gram-positive cells, in which the stain is primary
retained and Gram-negative cells, in which the primary stain is lost. Gram stain is one of the
most frequently performed laboratory diagnostic procedures. Gram stain interpretation gives
immediate information regarding the presence or absence of bacterial infection. This can be
ground for hospitalization and initiation of treatment and also initial choice of antibiotic
therapy. However, the competency of clinical laboratories is not well known in this study
area. Therefore, this study willdesigned to determine the gram stain competency and
associated factors in clinical laboratories in Mekelle. This study will be identified the gap on
laboratory professional as well as laboratory department on Gram stain examination and
interpretation and provide scientific evidence to responsible bodies for continues professional
and laboratory improvement.
Procedures:In order to undertake the above-mentioned study, theoretical examination
questions related with the topic; socio demographic characteristics, seven Gram stained panel
slides examination and interpretation will be taken from each study participants for analysis.
Written consent wills obtain from each study participants and they will be kindly asked to
give required information related with the study. The analysis of the data will be done by
principalinvestigator.
Time Allowed: You should grade and interpret one slide within five minutes. You will be
also provided 15 structured knowledge questions and background questionnaires to be
answered and filled within 30 minutes.

35
Risks and discomforts: There will have not any possible risks during examination and
interpretation of panel slides and answering of exam questions. It might be a possibility of
discomfort during examination and interpretation of panel slides and answering of exam
questions. Please feel free to ask which is not comfortable to you.
Benefits and Compensation: Participants in this study will be got 100 birr for lunch.
Confidentiality: The information obtained during this study will be remained confidential.
Disclosure of any of the data to third parties other than those allowed in the informed consent will
not permit. Records will remain confidential. To maintain confidentiality, the investigator will
keep records in locked cabinets and the results will be coded to prevent identification of the
volunteers. However, you can find out the competence results of your own.
Right to refuse or withdraw: I assure them that, they will be free to withdraw from the study at
any time and that they will be not discriminated in any form for education or other services.
Whom to Contact: The following contact addresses will be given to contact investigators at any
time.
Name of investigator: FikaduMirutsPhone No: +251914210892
Email: [email protected]
Department of Medical Laboratory Sciences, CHS, AAU, Tel. 0112 75 51 70
Sponsor Address: Mekelle University
Name of advisors: FatumaHassen (BA, MPH, PhD Candidate, AAU)
HabtamuMolla(Bsc, MSc,PhD Candidate, AAU)

36
Annex II: Informed Consent form (English Version)
I am informed fully in the language I understand about the aim of above mentioned research.
I understood the purpose of the study entitled with “assessment of Gram Stain competency
and associated factors in clinical laboratories health facility in Mekelle City, Ethiopia”. I
have been informed that I will be done examination and interpretation of gram stain and also
written examination. In addition I have been told all the information collected throughout the
research process will be kept confidential. I understood my current and future medical
services and others will not be affected if I refused to participate or with draw from the study.
Agree_______ Not agree_______
Therefore I give my consent freely for my participation in this study.
Code of participant: _____________________________
Signature:_____________ Date____________________
Name of researcher:____________________________________Signature:______________
Date__________________________

37
Annex III: Data collection tool for assessment of gram stain competency
and associated factors among laboratory personnel in clinical laboratory at
Mekelle city health facility.

Part 1: socio demographic data

1. Participant Code ------------------------------
2. Health facility type: 1. Government 2. Private 3.Uniformed

Socio demographic characteristics

NO Question Response
1 Age
2 Gender 1. Male 2. Female
3 Educational level 1. Certificate 2. Diploma 3. First Degree
4. Masters
4 Where did you graduate? 1. Government collage 2. Government
university 3. Private collage
4. Private university 5. Other specify
5 Type of microscope
6 Employment status (full time , part time)
7 Service year as clinical laboratory
professional
8 Are you participate in EQA program( 1. Yes 2. No
proficiency testing ) related to gram stain
9 Current accreditation status of your health
facility (not participate, `0` star , `1` star ,
`2` star ,`3` star, `4` star, `5` star)

10 Did you get trainingcertificate related to 2. Yes 2. No

gram stain?
If Yes on Q10, how many?
11 Did you get regular competency 1. Yes 2. No
assessment in your laboratory related to
gram stain
12 Are there health information resources 1. Yes 2. No
available in your work area

38
 Part 2: Gram stained panel slides report form for gram stain competency
assessment

Participant Code: -----------------------

To be filled by Laboratory personnel To be filled by Principal Investigator

Result
Slide Gram rxn Morphology No. of Other Expected Error
Result Remark
Number bacteria cells Type

GSS……..1

GSS……..2

GSS……..3

GSS……..4

GSS……..5

GSS……..6

GSS……..7

39
Part 3: Competence assessment for theoretical bases in Gram staining for
medical laboratory professionals working in Mekell city health institute, Mekelle,
Ethiopia

Q 1. What is Gram stain :

----------------------------------------------------------------------------------------------------------------
Q 2. Write one gram positive bacteria
----------------------------------------------------------------------------------------------------------------
Q 3. What is the difference between cell structure of gram positive and gram negative
bacteria
Q 4. Why is the decolourization important in gram staining?
----------------------------------------------------------------------------------------------------------------
Q 5.Write one possible cause of false gram reaction results?
----------------------------------------------------------------------------------------------------------------

Q 6. Using gram staining method, what is the appearance of Gram positive bacteria?
-------------------------------------------------------------------------------------------------
Q 7. Using gram staining method, what is the appearance of Gram negative bacteria :
------------------------------------------------------------------------------------------------------
Q 8. Which bacterias are decolorized by Acetone alcohol & containa counter stain colour
----------------------------------------------------------------------------------------------------------
Q 9. which bacteria resist decolourization& retain the color of the primary stain (Crystal
violet)?…………………………………………………………………………………..
Q 10. What is acounter stain in Gram stain technique? .........................................................
Q 11. What is a primary stain in Gram stain technique?...........................................................
Q 12. Write one possible morphologies of bacteria: …………...........................................
Q 13. what would happen if iodine were missed during gram staining ?
----------------------------------------------------------------------------------------------------------------
Q 14. What is the order of reagents used in the Gram stain?
--------------------------------------------------------------------------------------------------------
Q.15. Why we use heat fixative after smearing the sample before we use reagent to stain
bacteria?

----------------------------------------------------------------------------------------------------------------

40
Q.16. Write typical morphology of staphylococcus aures ?

Q.17.Why is the gram stain considered a differential stain?

----------------------------------------------------------------------------------------------------------------

Q.18.What does gram positive cocci in pairs mean(suggestion/interpretation)?

--------------------------------------------------------------------------------------------------------

Q.19.What does gram positive cocci in cluster mean?

----------------------------------------------------------------------------------------------------------------

Q.20.Why is it important to know gram positive or gram negative ?

----------------------------------------------------------------------------------------------------------------

Q. 21 Write a counter stain other than safranin be used in gram staining………………………

Q. 22 Write a primary stain other than crystal violet be used in gram staining…………….....

Q. 23 What happens if you reverse crystal violet and safranin stains ?

Q. 24 Is safranin basic or Acidic dye ?

Q. 25 Write one gram negative bacteria ?

41
Annex IV: Media preparation for blood agar

Purpose:Blood Agar (BA) isenriched medium used to culture those bacteria or microbes that do
not grow easily. Such bacteria are called “fastidious” as they demand a special, enriched
nutritional environment as compared to the routine bacteria. Blood Agar is used to grow a wide
range of pathogens particularly those that are more difficult to grow such as Haemophilus
influenza, Streptococcus pneumoniaandNeisseria species. It is also required to detect and
differentiate haemolytic bacteria, especially Streptococcus species. It is also a differential media
in allowing the detection of hemolysis (destroying the RBC) by cytolytic toxins secreted by some
bacteria, such as certain strains of Bacillus,Streptococcus, Enterococcus, Staphylococcus, and
Aerococcus.

Principle: Several species of Gram-positive cocci produce exotoxins called hemolysins able to
destroy red blood cells (RBCs) and haemoglobin. Blood agar, which is a mixture of tryptic soy
agar and sheep blood, allows differentiation of bacteria based on their ability to hemolyze RBCs.
Procedure
1. Add 40 gm Blood agar powder in 1000 ml of distilled water
3. Heat until completely dissolved on the water boil
4. Autoclave at 121 0c for 15 minutes
6. Cool until 45-50 0c
7. Aseptically add sheep blood 5% (7%) and mix well
8. Dispense 15-20 ml of the ready media on to the petridish
9. Wait until solidify or Dry
10. Check sterility at 37 0c incubators for 24 hours with positive control
11. Inoculate or subculture
12. Check growth within 24 -48 hours

42
Annex V: Gram stain smear preparation procedure
1. Labelling slides: - Every slide must be labelled clearly with codes by a lead pencil
2. Smears should be spread evenly covering an area of about 15–20 mm diameter on a slide.
3. After making a smear, leave the slide in a safe place for the smear to air-dry, protected from
dust, flies, ants, and direct sunlight.
4. To fix the smear, rapidly pass the slide; smear uppermost, three times through the flame of a
spirit lamp or pilot flame of a Bunsen burner.

5. Allow the smear to cool before staining it.

Note: our study used six slides prepared from known bacterial strains (ATCC) and one slide
prepared from patient sample. The ATCC was first took from Deep freezer and allow to room
temperature until become warm. Then it was subculture on blood agar and after 24 hours, the
smear was done from growth culture after dilution. To get different grading smear, tenfold serial
dilution by normal saline could be conducted. I took one colony from subcultured blood agar by
using sterile wire loop then add the colony in 50ml normal saline containing test tube(tube 1) and
then vortex in order to emulsify. We do not know the number of bacteria that are present in test
tube one ,so I prepared additional 5 test tube that are contain 450ml of normal saline in order to
conduct 10 fold serial dilutionthenTo prepare slide smear with few number of bacteria by using
micro pipette transfer 1 ul from test tube 2 or 3, for moderate from test tube 4 and for few from
test tube 5 this grade was confirmed by Experts those are present at ayder comprehensive
specialized hospital microbiology department. Slides coded as GSS (Gram Stain Slide) 1, GSS 2,
GSS 3, GSS 4, GSS 5, GSS 6 and GSS 7. GSS 1 prepared frompus patient sample with no
microorganism and many pus cells,GSS 2 prepared from S.aurous, GSS 3 prepared from
bacillus,GSS 4 prepared from N. gonorrhoeae, GSS 5 slides prepared from E.coli. , GSS 6
prepared from S.pyogen and GSS 7 prepared from S, pneumonia.

43
Annex VI: Gram Stain Procedure
Principle
The Gram stain, a differential stain was developed by Hans Christian Gram, a Danish physician,
in 1884. Gram staining classifies bacteria into 2 major groups, Gram positive and Gram negative
bacteria. The Gram stain reaction is based on the difference in the chemical and physical
composition of bacterial cell wall. Gram positive cells have a thick peptidoglycan layer, whereas
peptidoglycan layer in Gram negative cells is much thinner and surrounded by outer lipid
containing layer.
In Gram negative, the higher amount of lipid in the formation of large pores thus facilitating the
leakage of crystal violet-iodine complex and resulting in the decolonization of the bacterium
which later takes this complex counter stain. In contrast, the Gram positive cell wall are thick and
composed mainly of proteins and cross linked mucopeptide, when treated with alcohol it causes
dehydration and closure of the cell wall pores thereby not allowing the loss of complex and cell
retains primary stain.
1. Fix the dried smear.
2 Cover the fixed smear with crystal violet stain for 30–60 seconds.
3 Rapidly wash off the stain with clean water.
Note: When the tap water is not clean, use filtered water or clean boiled rainwater.
4 Tip of all the water, and cover the smear with Gram’s iodine for 30–60 seconds.
5 Wash off the iodine with clean water.
6. Decolorize rapidly (15 -30s) with acetone–alcohol. Wash immediately with clean water.
7. Cover the smear with Safranin for 30 sec to 1 min.
8. Wash off the stain with clean water.
9. Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry.
10 Examine the smear microscopically, first with the 40 × objective to check the staining and to
See the distribution of material, and then with the oil immersion objective to report the bacteria
and cells.

44
አባሪ(Annex) VII : የስምምነት ቅጽ(Consent form) የአማርኛው ቅጂ(Amharic version)
ሙለለሙለ በምረዳው ቋንቋ ከፍ ብሎ ስለተመለከተው ጥናታዊ ስራ አላማ መረጃዎች ተሰጥቶኛል፡፡ርእሱ
“በመቐለኢትዮጵያበሚገኙየህክምናተቋማትውስጥየሚሰሩየህክምናላብራቶሪባለሙያዎችናየላቦራቶሪክፍልበግራምእ
ስቴይንምርመራላይያላቸውብቃትመመዘን”የተሰኘውንጥናትአላማተረድቼዋለሁ፡፡ምርመራእናስለክፍሉኣንዳንድመረ
ጃእንደሚደረግተነግሮኛልእንዱሁምየጹሁፍምርመራምእንደሚደረግተነግሮኛል፡፡በተጨማሪምበጥናትሂደትውስጥበ
ሙለየተሰበሰቡመረጃዎችንሁሉሚስጥራዊበመሆንእንደሚጠበቁተነግሮኛል፡፡የወቅቱእናየወድፊትየህክምናአገሌግልቶ
ችከጥናቱበወጣወይምበመሳተፍፍላጎቱባይኖረኝተጽኖእንደማይደርስብኝተረደቻለሁ፡፡
እስማማለሁ ________ አልስማማም ________
ስለዚህበዚህጥናትላይእንድሳተፍበነጻነትፍቃድንሰጥቻለሁ፡፡
የተሳታፊውኮድ፡ ________________
ፊርማ፡ __________ ቀን፡ ________________________
የተመራማሪውስም፡ ____________________ ፊርማ፡ ________________
ቀን፡ ________________________

45
አባሪ( Annex)VIII: የመረጃ ቅጽ(Information Sheet) የአማርኛው ቅጂ(Amharic
Version)
የፕሮጀክቱርእስ፡በመቐለኢትዮጵያበሚገኙየህክምናተቋማትውስጥየሚሰሩየህክምናሊብራቶሪባለሙያዎችናየላቦራቶሪ
ክፍልበግራምእስቴይንምርመራላይያላቸውብቃትመመዘን”
የዋነኛ ተመራማሪው ስም፡ፍቃዱ ሙሩፅ ተስማ
ተቋም፡አዱስ አበባ ዩኒቨርስቲ
የእስፖንሰሩ ስም፡መቐለ ዩኒቨርስቲ
ይሄየመረጃቅጽከፍብሎበርእሱበተመለከተውፕሮጀክትውስጥተሳታፊለሚሆኑየህክምናሊብራቶሪባለሙያዎችየተዘጋጀ
ነው፡፡በበጎፍቃደኝነትእንዲሳተፉበመጠየቅእናበጥናቱስለተከናወነውሙለሂደትየምንነግራችሁይሆናሌ፡፡
መግለጫ እና የጥናቱ አላማ
ግራምስቴይንባክቴሪያንበሁለትክፍልችለመለያየትአሁንምበከፍተኛሁኔታበንቃትጥቅምላይእየዋለያለክላሲክባይልጂካ
ሌአካሄዴነው፡ግራምፖዘቲቭሴሎችቀለምበዋነኛነትተይዞባቸውየሚቆይሲሆንግራምኔጌቲቭሴሎችዋነኛቀለማቸውየጠ
ፉናቸው፡፡ግራምስቴይንአንደበተዯጋጋሚሁኔታየሚከናወንየሊብራቶሪምርመራበቅደምተከተልነው፡፡የግረምስቴይንት
ርጓሜየባክቴሪያቁስሇትንመኖርወይምአሇመኖርበተመሇከተእናበቁስሇትሂዯትሊይተሳታፊየሆኑየባክቴሪያሞርኮታ
ይፕስሊይፈጣንየሆነመረጃንይሰጣሌ፡፡ይሄምወደሆስፒታልገብቶለመታከምእናህክምናለማስጀመርእንደመሰረትሆኖ
ሊያገለግልየሚችልሲሆንእንዱሁምየአንቲባዮቲክህክምናንየመነሻምርጫሉሆንይችላል፡፡ይሁንእናበዚህየጥናትአካባቢየ
ህክምናላብራቶሪባለሙያዎችብቃትበሚገባአይታወቅም፡፡በመቐለኢትዮጵያበሚገኙየህክምናተቋማትውስጥየሚሰሩየ
ህክምናሊብራቶሪባለሙያዎችናየላቦራቶሪክፍልበግራምእስቴይንምርመራላይያላቸውብቃትለመወሰንየተነደፈነው፡፡
የአካሄድ ቅደም ተከተል
ከፍብሎየተመለከተውንጥናትለማከናወንከርእሱጋርተጓዳኝየሆኑየተወሰኑየምርመራጥያቄዎች፣ሶሾዱሞግራፊክባህሪያ
ቶች፣ባለፓኔልእስላይድምርመራእናትርጓሜለምርመራከእያንዲንዱየጥናትተሳታፊዎችየሚወሰድይሆናል፡፡የጹሁፍስም
ምነትከእያንዲንደየጥናቱተሳታፊዎችተወስድተገቢውንከጥናቱጋርየተገናኙመረጃዎችእንዱሰጡይጠየቃለ፡፡የመረጃው
ትንተናበተመራማሪውየሚደረግይሆናል፡፡

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አደጋዎችእናተግዲሮቶች፡በምርመራውወቅትእናበፓኔልእስላይዶቹትግበራወቅትእንዱሁምየምርመራጥያቄዎችመልስ
በሚሰጡወቅትምንምአይነትአደጋዎችወይምተግዲሮቶችየማይኖሩይሆናል፡፡
ሚስጥራዊነት፡በዚህጥናትወቅትየተገኙመረጃዎችሚስጥራዊእንደሆኑይቀጥላሉ፡፡መረጃከተሰጠበትስምምነትውስጥከ
ተፈቀደላቸውአካላትበስተቀርለማናቸውምሶስተኛወገኖችመረጃይፋማዴረግአይፈቀድም፡፡ሪከርድችሚስጥራዊእንደሆ
ኑይቀጥላሉ፡፡ሚስጥራዊነትንለመጠበቅተመራማሪውሪከርድቹንበሚቆለፉመደርደሪያዎችውስጥየሚያስቀምጣቸውሲ
ሆንውጤቶችምበበጎፍቃደኞችመለየትእንዲይችለበኮድምልክትይደረግባቸዋል፡፡የሚተዉመብቶች፡ለእነርሱከጥናቱበ
ማንኛውምጊዜመውጣትእንደሚችሉነጻነቱእንዳላቸውእንዱሁምለትምህርትወይምለሌላአገልግሎቶችመገለልእንደማ
ይደረግባቸውአረጋግጬሊቸዋለሁ፡፡
ማንንማግኘትይፍልጋሉ፡፡የሚከተለትየግንኙነትአዴራሻዎችበማንኛውምጊዜተመራማሪውንማግኘትይችላሉ፡፡
የተመራማሪው ስም፡ፍቃዱ ሙሩፅ ተስማ ስሌክቁጥር ፡09 14 21 08 92
ኢሜይሌ፡ [email protected]
የአማካሪው ስም፡ፋቱማ ሃሰን (ቢኤስሲ፣ኤምኤስሲ፣ፒኤችዱእጩ፣አአዩ)
ሃብታሙሞላ( ቢኤስሲ፣ኤምኤስሲ፣ፒኤችዱ;እጩአአዩ)

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Annex IX: Declaration
Title of Project: Assessment Gram stain Competency and associated factor in Clinical
Laboratories in Mekelle city health institute, Ethiopia
I, the undersigned, declare that this MSc research project is my original work. It has not been
presented for a degree in any other University. False statements could be cause for
invalidating this research project and may lead to other administrative or legal action.
Principal investigator: Fikadu Miruts
Address: Department of Medical Laboratory Sciences, AAU
Signature: _____________________ Date: ___________________
Advisors: FatumaHassen (BA,BSc, MPH, PhD Candidate)
HabtamuMolla(BSc,MSc, phD Candidate)
Address: Department of Medical Laboratory Sciences, AAU
Signature: _____________________ Date: __________________

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