 NOTE: Chromosomes in the bacterium are
UNIT 2 fitted in the nucleoid.
TOPICS OUTLINE
Bacterial Genetics
Bacterial Growth and Nutrition
Bacterial Metabolism
TOPIC 1: BACTERIAL GENETICS
➔ The inherited traits of microbes include their
shape and structural features, metabolism,
ability to move or behave in various ways
and ability to interact with other organisms-
perhaps causing disease.
➔ The ultimate aim of a cell is to produce
proteins that are responsible for cellular
structure and function and to transmit the
information for accomplishing this to the next
generation of cells.
Chromosome  Consist of a single, closed,
circular piece of dsDNA that is
super coiled to fit inside the
cell.
Plasmids
 Extrachromosomal dsDNA,
contains extra information on
small circular pieces.
 Genes not essential for
bacterial growth, so they can
be gained or lost
 code for antimicrobial
resistance (And sometimes
toxins or other virulence
factors).
Genes  Segments of DNA of an
organism.
Genotype
 Genetic potential of DNA of an
organism.
 All the characteristics that are
coded for in the DNA
bacterium and that have the
potential to be expressed.
 Constitutive genes Vs. Silent
genes (inducible)
Phenotype  Observed characteristics
expressed by the genome
 NOTE: A genome of a bacterium is normally
composed of: Chromosome + Plasmid.
 NOTE: DNA contains segments of genes
that code for functional produc
 NOTE: Constitutive genes are being
replicated & transcribed to be manifested as
characteristics of a bacterium. (E.g. Genes
that code for protein that is a component of
a flagella/cell wall.)
 NOTE: Silent genes are not usually
expressed, but can be induced in certain
circumstances when the cell needs it in
times of stress.
 NOTE: Genotype & Phenotype are the
parameters that can be used to identify and
classify a bacterium. (E.g. bakit may pili ang
bacteria, bakit may makapal na cell ang
gram + na bacteria)
➔ DNA of a bacteria is important for bacterial
growth.
➔ One of the most important concepts in
bacterial genetics is the process of bacterial
recombination.
➔ The reason why we have bacteria of the
same species or other species being able to
elaborate or express toxins being found in
different bacterial species is the process of
gene transfer and recombination.
RECOMBINATION
➔ is the incorporation of the donor DNA
(foreign DNA: can be from the same species
of bacteria or other species of bacteria) to a
recipient DNA giving birth to relatively new
whole DNA.
➔ Recent application of recombination: the
creation of COVID-19 Vaccine such as
Astrazeneca (RNA Vaccine), with the use of
bacteriophage that infects a bacteria to
reproduce the vaccine.
➔ Recombination also naturally occurs in
bacteria.
 coming from age. A from one
the donor bacteriopha bacteria to
cell upon ge infects another
lysis of the the donor
donor cell. cells and  There
along the should be a
 The free process of cell to cell
DNA will viral contact and
have replication, sex pili.
recombined these
the bacteriopha  In the
chromosom ges illustration
al DNA of incorporate below,
the recipient to their conjugation
bacterial genome can be
cell. segments of means of
DNA coming gene
 can be an from the transfer
avenue for bacterial involving
the host itself chromosom
developmen e or plasmid
t of antibiotic  Upon lysis, (in other
resistant these books we
and bacteriopha can also
developmen ges are add
t of released transposons
virulence from the or the
factor of the donor cells/ jumping
mentioned host cell. genes)
bacteria These
above bacteriopha
ges would
infect
another
bacterium

 No cell to  No cell to  Requires

cell contact cell contact cell to cell
because the because of contact.
donor cell
the
lyse before
the recipient bacteriopha
cell gets the ge..
gene.
 Competent Sex pilus- use in
bacteria-  Bacterioph qthe conjugation
we call the age- of bacterias
recipient cell bacteriopha
ge is a virus
to be a
capable of
competent infecting
GENE TRASNFER
 transfer of a donor DNA from a bacterium to bacteria if it bacteria.
another bacterium(whether same species or not) has the
so that their genes will combine to produce a ability to  Temperate
relatively new set of genetic material. form phage-
recombinant natural
3 MECHANISMS OF GENE TRANSFER vectors for
DNA via the
gene
mechanism transmission
Transformation Transduction Conjugation
of among
transformati bacteria
 Recipient  It has an  nearest way on.
cell agent of the of microbe Haemophilus,
assimilate/ activity to have a Streptococcus  Corynebact
partake the called gene and Neisseria erium
free DNA bacterioph transferred diphtheria
 which METABOLISM
causes the  is the summary of all the chemical reactions
disease of an organism/bacterium
Diphtheria
(involves CATABOLIC ANABOLIC REACTION
respiratory REACTION
tract and
heart).  degrade substance  building up more
substrate/ complex complex substances
 The substances to from building blocks
Corynebact simpler ones, to or simpler
erium have a surplus of molecules
diphtheria energy
that is
usually  Does not require  Requires ATP
subject to energy but releases energy.
bacteriopha energy.
ge infection
carries the  Requires oxygen.  Does not require
gene that oxygen.
codes for its
toxins called  Ex: Hydrolytic  Ex: Amino acid/
lysogeny. reactions: digestion nucleic acids
becomes DNA or
RNA
TRANSFORMATION  Sugar monomers
becomes more
complex/ structures
necessary for cell
wall or capsule for a
bacterium.

TRANSDUCTION AMPHIBOLIC REACTION

 like the Kreb’s cycle (tricarboxylic cycle), plays
an anabolic and catabolic role in the bacterial
metabolism
 bridges anabolic and catabolic reactions (accdg
to other books)
CONJUGATION
 NOTE: Ang difference lang between
chromosome transferal and plasma transfer
is their final morphology or configuration of
the recipient bacterium.

 NOTE: The presence of a certain pathway

PLASMID TRANSFER
 (the gene that is contained in the plasmid does
not incorporate the chromosomal DNA).
(be it anabolic or catabolic) depends on the
presence of the enzyme (6 types of enzyme)
that catalyzes the reaction.

 NOTE: peritrichous/monootrichous -flagella

ang pinag-uusapan not pili/ sex pilus.
 NOTE: In the laboratory, we also take
advantage of these chemical reactions for
 NOTE: Do not forget the prototype bacteria/
the identification of organisms or bacterium.
bacterium for each mechanism of gene
transfer and recombination.
 NOTE: A bacterium differs in terms of their
enzyme profile. hence the different
 reactions they are capable of doing. So that,
ENTNER-DOUDOROFF PATHWAY
their end products differ also. These end
products of metabolism are the ones ● Converts glucose-6-phosphate( rather
detected in the laboratory, usually the color than glucose) to pyruvate and
change of chemicals or indicator chemicals. glyceraldehyde phosphate, which can be
This is the importance of understanding the funneled into other pathways
reactions possible for a given bacteria/ ● Generates one NADPH per molecule of
bacterium/ microorganism. glucose but uses one ATP
● Aerobic process used by
Pseudomonas,Alcaligenes,
 NOTE: Microbiologists use these metabolic Enterococcus fecalis and other bacteria
differences as phenotypic markers in the lacking certain glycolytic enzymes.
identification of bacteria.

 NOTE: All three pathways have the common

DIAGNOSTIC SCHEMES ANALYZE EACH end product which is the pyruvate but uses
UNKNOWN MICROORGANISM FOR: different substrates.
 NOTE: All three pathways are catabolic
reactions.
 utilization of various substrates as a carbon
source,

 production of specific end products from PATHWAY SUBSTRATE

various substrates, and
EMP GLUCOSE
 production of an acid or alkaline pH in the
test medium. PPP GLUCOSE but would
also yield precursors for
THREE MAJOR METABOLIC PATHWAYS nucleic acid synthesis

EMP GLYCOLYTIC PATHWAY

EDP GLUCOSE-6-
● Major pathway in conversion of glucose PHOSPHATE
to pyruvate
● Generates reducing power in the form of
NADH2 DIAGRAM OF PENTOSE PHOSPHATE, TCA
● Generates energy in the form of ATP CYCL
● Anaerobic, does not require oxygen
● Used by many bacteria including all
members of Enterobacteriaceae

PENTOSE
PHOSPHATE(PHOSPHOGLUCONATE)
PATHWAY

● Alternative to EMP pathway for

carbohydrate metabolism
● Conversion of glucose to ribulose-5-
phosphate which is rearranged into other
3-,4-,5-,6-,7-, carbon sugars
● Provides pentoses synthesis
● Produces glyceraldehyde-3-phosphate
which can be converted to pyruvate
● Generates NADPH which provides E AND EMP PATHWAY
reducing power for biosynthetic  Red - Substrates/intermediary substances
reactions highlighted in red are precursor molecules
● May be used to generate ATP(yield is or metabolites.
less than in EMP)  They are precursors or building blocks that can
● Used by heterolactic fermenting bacteria be used to create a more complex structure in
such as Lactobacilli and by Brucella the bacterial cell through the different pathways.
abortus which lacks some enzymes
needed required in EMP pathway
 Green - Reducing substances necessary for
• 3-Phosphoglycerate
the oxidative phosphorylation and electron
• Phosphoenolpyruvate
transport chain in producing ATP.
• Pyruvate
 Ex. NADH, FADH, NADPH- important for
• Acetyl CoA
production of ATP necessary for fueling for
• Ketoglutarate
example, protein channels in the plasma
• Succinyl CoA
membrane.
• Oxaloacetate
 Ex. Sodium potassium pump, sodium
bicarbonate pump to have balance between the
inside and outside environment of the cell.
NUTRIENTS
 Phosphate - produced at the substrate level • Gases Carbon dioxide (CO2)
phosphorylation. • Oxygen (O2)
• Ammonia (NH3)
SUBSTRATE LEVEL PHOSPHORYLATION • Organic compounds, including amino acids
 In substrate-level phosphorylation, high-energy • Water (H2O)
phosphate bonds produced by the central • Nitrate (NO3-)
pathways are donated to adenosine • Phosphate (PO43-)
diphosphate (ADP) to form ATP directly from • Hydrogen sulfide (H2S)
the substrate as opposed to generation via the • Sulfate (SO42-)
electron transport chain. • Potassium (K+)
 Dependent on the type of bacterium if aerobic • Magnesium (Mg2+)
• Calcium (Ca2+)
or non-aerobic if capable of fermentation or
• Sodium (Na+)
respiration also known as oxidation. • Iron (Fe3+) Organic iron complexes
 But there are also bacteria which are capable of
doing both in producing intermediary precursor Why are we studying these concepts?
 Metabolism is important for the growth of
molecules and atp ex. Facultative anaerobe
bacteria and its survival.
 Understanding metabolism is important for
 Metabolism of microorganisms are important for
Medical Technologists to better appreciate the
cell structure and function. At the same time in biochemical tests done incorporated as
the provision of ATP or the maintenance of methods of identification of bacteria in
homeostasis or equilibrium inside the cell laboratory.
relative to the outside environment.  A knowledge of which mechanisms bacteria
use to generate ATP is important for designing
 For a cell to not lyse, we need an ATP needed laboratory protocols for cultivating and
that is generated through the electron transport identifying these organisms. For example,
some bacteria depend solely on aerobic
chain present in the plasma membrane of the
respiration and are unable to grow in the
bacterium. We need ATP for the cell to absence of oxygen (strictly aerobic bacteria).
maintain its integrity. Others can use either aerobic respiration or
fermentation, depending on the availability of
oxygen (facultative anaerobic bacteria). For
still others, oxygen is absolutely toxic (strictly
anaerobic bacteria).

 NOTE: Bacteria reproduce by a process

called binary fission.

 NOTE: Bacteria growth do not only refer to

bacteria but the overall bacterial population
itself.

PRECURSOR METABOLITES

• Glucose 6-phosphate 4 PHASES OF BACTERIAL GROWTH

• Fructose 6-phosphate
• Pentose 5-phosphate
• Erythrose 4-phosphate
  QUESTION: may mga existing ba na bacteria
na extinct na ngayon?
 ANSWER: Thiomargarita magnifica (not sure
kung meron pa)

COMPUTATION
FORMULA:

Generation Time = t/n

Where: t = Time interval
n= number of generation
LAG PHASE
 Is the maturation of the bacterium Different generation time of bacteria
Preparation of bacterial structures for replication  E.coli = 17- 20 minutes
LOG PHASE
 The drastic or rapid multiplication of bacteria EXERCISE 1:
Isang E. coli, you cultured it and then after 3
STATIONARY PHASE
hours and 20 mins. Ilan yung inneexpect mong
 An equal birth and death rate of bacteria is colony forming units?
observed.
DEATH PHASE Given: 3 hrs 20 mins (200 minutes)
 Increase in toxic by products produced by Reproduce every 20 minutes
bacterial growth which in turn kills bacteria Doubling time = 2
itself.
# of generation = 200/20 = 10
= 2^10
= 1,024 bacteria
Exercise 2:
GENERATION TIME TABLE After 3 hrs and 20 mins of inoculating 100 E.coli.
How many bacteria are you expecting

#
of generation = 200/20 = 10
= 2^10
= 1,024
=1,024 x 100
= 102, 400 bacteria

BACTERIAL GROWTH AND NUTRITION

The requirements for bacterial growth can be
divided into two categories:

1. Physical requirement - include temperature,

pH, and osmotic pressure
2. Chemical requirement -include sources of
carbon, nitrogen, sulfur, phosphorous,
oxygen, trace elements, and organic growth
factors.

TEMPERATURE REQUIREMENTS
Psychrophile/Psychotrophs  optimal
temperature
of 10°C-
20°C
Mesophiles  optimal
 Sa food industry, kailangan nila ng maraming
temperature
mikrobyo yung may vigor pa, yung nasa lag
of 20°C-
phase pa muna para magmumultiply ng
40°C
maramihan at macatalyze ng reaction na
Thermophiles-  optimal
important sa creation ng beer/cheese/yogurt.
 temperature BACTERIAL PH AND OSMOTIC PRESSURE
50°C-60°C OSMOTIC PRESSURE
Hyperthermophiles  optimal  It is the pressure exerted inside the cell
temperature because of the movement of water molecules
of 75-80°C from low solute concentration to high solute
to 95-100°C concentration area.
 The plasma membrane as well as cell wall
TEMPERATURE VS RATE OF GROWTH provides an envelope for the maintenance of
 The peak of the graph represents at which the the structural integrity of the cell because there
rate of growth is at maximum. is a high pressure inside the cell for it to not
burst.
 NOTE: Optimum temperature is usually  Another concept for us to understand the
expressed in range of temperature. concept of osmotic pressure is the addition of
salt to water.
EXAMPLE:  Hypertonic solution - solution with a high
Mesophiles where most of the pathogenic bacteria solute concentration as compared to a solvent.
are. The optimum temperature for mesophiles is  Hypotonic solution -solution with a high solute
20°C to 40°C. concentration as compared to a solvent.
 Isotonic solution - solution that has the same
This is where most pathogenic bacteria are salt concentration as cells and blood. Example
classified because the body temperature is at is saline solution.
36.5°C-37.5°C.

RATE OF GROWTH
 Left extremity of the graph up to the peak of the
graph.
 No stationary phase. Everything is constant.
 Few microorganisms are pathogenic but usually
can be grown at higher or lower temp relative to
the mesophilic range of temperature.

EXAMPLE:
PSEUDOMONAS AERUGINOSA
 Microbe that cannot be killed by safeguard. Can
thrive and multiply on your soap.
 Obligate anaerobe gram negative bacilli
 Can cause culminant or localized infection
 Optimal temperature for growth is at mesophilic
range 35°C-37°C. But can also thrive at
temperatures of 42

LISTERIA MONOCYTOGENES
 Mesophilic bacterium common cause of
neonatal infection or septicemia.
 Causes infection to a woman who is pregnant
and later on transmit infection to the child or to
the fetus.
 This bacteria can grow and survive at
temperature 4°C to 8°C (ref temp).
 Normally contracted at ingestion of
unpasteurized milk
Room temperature- 22°C- 24°C
Ref temperature- 2°C- 6°C

 NOTE: When we culture bacteria at lab,

ideally there are no contaminants pero
minsan ang nangyayari may mga fungal
elements na nabubuo kasi fungal elements
can grow at body temperature din. Kaya
ideally dapat separated ang incubator niyo
for bacte and fungi microorganisms.

 Bacteria are 80-90% water
 Osmotic substances- eg. salt and sugar
 eg. of pathogenic halophiles: Vibrio cholerae, V.
parahaemolyticus, Bacillus anthracis
 When we say osmotic substances, these can
be any substances that can trigger the
movement of water molecules along the
gradient, so it can be from water molecules
inside the cell going outside the cell making it
shrink.
 Under these concepts the term halophiles
emerged ( Halogens, salts from the group of
elements like Fluoride, Bromide, etc.)
 In the presence of salt, most of the pathogenic
bacteria do not survive except for pathogenic
halophiles
 Pathogenic Halophiles can thrive in relatively
high concentration of salt , we also have
staphylococcus species which can tolerate
relatively high concentration of salt but not as
high as the salt concentration being tolerated
by Vibrio and Bacillus. That's why we have the
Mannitol Salt Agar medium to our cultivating or
growing of Staphylococcus species.
 NOTE: Remember that bacterias are 80-90%
water. Imagine if you expose the bacteria in a
hypertonic saline ay lalabas lahat ng 90 ml
water out of 100 ml volume so the cell wil
plasmolyzed , the cytoplasm is plasmolyzed.
 NOTE: pH is one of the physical factors that is
very important for bacterial growth. pH can be
acidic or alkaline,
 Using the Henderson-Hasselbalch equation, we
also have buffer solutions. The presence of
buffer solution stabilises or maintain an
equilibrium between the amount of the different
ions in the solutions
 In culture medium we incorporate buffers so
that the growth of the bacteria a in relation to
the bacterial growth curve; In the stationary
phase- there is stationary phase because of
acidic by-products namamatay yung mga
mikrobyo, but the presence of buffers like
peptones, amino acids, and phosphates
improves or there is a more optimal growth of
bacteria, but that is for growing bacteria.
 The by-products or acids produced by the
metabolism of nutrients by the bacteria is
advantageous when applied in bacterial
identification using pH indicator . We can
differentiate or detect the colour change in the
test system in order to identify if its positive for
that bacteria or not; that's the importance of
acids.
 Bacterial growth, bacteria usually thrive at near
neutral pH of 6.5-7.5 ( point narrow range ,
which is best support the growth of bacteria) we
also have outliers bacteria, there are bacterias
that can grow at lower acidic pH, classic
example is Helicobacter pylori( gram negative,
bacilli, non-spore forming, Urease Test)
How does H. pylori afford to live in the stomach
with a pH of <2, in some books 2-4?
 Such lower acid is because of the presence of
HCl produced by the parietal cells by the antral
part of the stomach. H.pylori can survive in
harsh conditions because of the enzyme
urease, the urease catalyses the conversion of
urea to ammonia, the ammonia is an alkaline
substance that would protect the H. pylori from
the acidic environment by neutralising the HCl
 H. pylori is the etiologic agent for peptic ulcer
disease. The risk factor for peptic ulcer is the
presence of H. pylori in the stomach, the use of
NSAIDs, and cigarette smoking. A significant
portion of the population really has H. pylori in
their stomach.
 Ions/metals/vitamins serves as cofactor for the
Ions/metals/vitamins serves as cofactor forthe
enzyme to be active.
 For cellular functions of plasma membrane,
efflux and influx should be maintained of ions
para ‘di magkaroon ng imbalance between the
interior and exterior environment of the cell.
 ATP is needed to perform cellular function.
Example: Ion pumps
 It can be used to Identify bacteria
1. Bacterium can use only glucose as a source
of carbon.
2. Bacterium can use both glucose and lactose
as a source of carbon.
 NOTE: Bacteria differ widely in their ability to
use different sources of these molecules.
Kung yung E.coli ay lactose fermenter, it means
it can also use glucose as source of precursor
metabolites and energy
 Microorganisms can be grouped according to
Chemotrophs, Phototrophs and further into
Chemoheterotrophs, chemoautotrophs,
photoheterotrophs and photoautotrophs
depending on the type of chemical that it uses
as carbon source
Heterotrophs
 almost always the type of micropathogenic
microorganism
 it uses organic compound as carbon source
Chemotrophs are also known as lithotrophs
AEROBES & ANAEROBES
Obligate aerobes
 Aerobes that lives only in an aerobic
environment
 Oxidation or respiration is a more efficient
mode of metabolism for producing energy and
metabolic substrates.
 The underlying reason is that there are these
enzymes called catalase and superoxide
dismutase that allow toxic forms of oxygen to
be neutralised.
 When oxygen is used as final electron acceptor
in oxidative phosphorylation in the Electron
transport chain, nagkakaroon ng reactive
oxygen species or free radicals (Toxic to
microbial growth)
 Due to the presence of enzymes catalase and
superoxide dismutase, Obligate aerobes can
ignore toxic forms or oxygen (Reactive oxygen
species and free radicals)
EXAMPLES:
Mycobacterium tuberculosis, Pseudomonas
aeruginosa
FACULTATIVE ANAEROBES
 Are aerobes that can grow in an anaerobic
environment but preferencially grow in the
presence of oxygen (Mas efficient kung may
oxygen)
 Grows best when oxygen is present
EXAMPLES:
Escherichia coli
OBLIGATE ANAEROBES
 Grow only in the absence of oxygen because
they lack enzymes (catalase and superoxide
dismutase) to neutralize the active forms of
oxygen (Di nila matolerate yung oxygen)
 These microorganisms don’t employ respiration
or oxidative pathway of reactions instead they
are the microorganisms that ferments
substrates
EXAMPLES:
Bacteroides fragilis (Usually found in colon or gut)
AEROTOLERANT ANAEROBES
 Anaerobes that grow preferentially in the
absence of oxygen, but continue to grow with
the presence of oxygen; but don’t use oxygen
in their metabolism
EXAMPLES:
Streptococcus spp. –normal flora of the human body
MICROAEROPHILES
 Aerobic organisms require low concentration of
oxygenCan grow at an oxygen atmospheric
pressure of 5-6%
 Note: Air is consist of 21% O2 and 1% CO2
 In the laboratory, the aerobic incubator has
10% CO2 and 18% O2 (Ideal setup for both
aerobic and facultative anaerobes growth)
 Gas concentration is expressed as partial
pressure
 Culture jar/candle jar or microaerohilic pockets
to produce 5-6% O2 environment
EXAMPLES:
Campylobacter jejuni (Usually resides in the small
intestine; usually sa mga lower vertebrates)
CAPNOPHILIC BACTERIA
 requires relatively higher amount of CO2 (5-10%)
to grow optimally
EXAMPLES:
Neiseria gonorrhoeae – causes gonorrhoeae
 NOTE: Dapat alam yung mga Representative
microorganisms for each type of bacteria in
context of oxygen requirement.
 BACTERIA CULTURE MEDIUM

3 GENERAL TYPES OF AGAR MEDIA

(2 in tubes, 1 in petri dish)
Forms of Agar media in tubes
Slant  Solid medium in a test tube.
Allowing the test tube to stand at
an angle then the agar solidifies
and it will assume a slanting
position. And the surface of the
agar will also be slanted.
Deep tube  The test tube is in a vertical
1. LIQUID position until the agar solidifies.
 broth (chicken/potato broth) And results to a flat surface above
 Pwede tubuan ng microbes the medium.
Slant butt  A combination where there’s a
2. SOLID
agar valley or plateau.
Forms of Agar media
Petri dish  Shallow cylindrical containers with
fitted lids that are designed
specifically for microbiology or cell
culture use.

 NOTE: Slant, Deep tube and Slant butt agar

is used in chemical reactions in IMVIC
AGAR reactions. A biochemical test in the
 (solidifying agent) is a derived from a complex
identification of a bacterium.
polysaccharide from a marine algae
 NOTE: IMVIC is a battery of biochemical
 superior to other solidifying agent as an tests. IMVIC stands for Indole, Methyl Red,
ingredient in making culture medium Voges Proskauer and Citrate.
 NOTE: Julius Richard Petri invented the
 ( most of bacteria do not degrade agar to use it
petri dish.
as a source of carbon)
 NOTE: Inoculum (e.g. nasopharyngeal swab
 it only acts as a BASE ( walang ginagawa and ( baka may resistance ka sa S. aureus)
does not provide nutrition)  NOTE: Culture (e.g Mannitol Salt agar)
 NOTE: Note: there is no gas medium
 LIQUEFIES: 100 degrees ( boiling point of
water and at sea level remains liquid until the Chemically defined Complex medium
temperature drops to about 40 degrees (it medium
solidifies)- this what makes it ideal  very exact  used in the lab
 we know the amount
and particular EXAMPLE:
ingredients nutrient agar (beef
comprising the extract that are not
medium chemically defined)
 used in growing
fastidious organisms
 microbiological
 assays
 not to detect or
quantify bacteria
 using bacteria
(reagent) to
measure an analyte

EXAMPLE:
Megaloblastic anemia-
B12 deficiency (used
Euglena gracilis
( because it needs B12
for growth) as a
reagent)

Result: B12 deficient:

blood is collected and
incorporated in the
medium, the E. gracilis
will not grow