Exam 1 Study Guide

This guide covers the first three chapters. You will sort the microbes into groups, make them visible, and then take a bacterial cell apart. Work one chapter at a time. Read the "what to know" prose, then cover the right side of each table and rebuild it from memory. Say the sequences out loud in order. Finish each chapter with its ten self-test items. The cram sheet at the end holds every name, number and step in one place.

Chapter 1The Microbial World and Why It Matters

What to know

A microbe is any living thing too small to see without a microscope. That one size rule gathers a mixed crowd. The organisms in it are not close relatives. So the first job is sorting them.

The first cut is cellular against acellular. Bacteria, archaea, fungi and protozoa are made of true cells. Helminths join the list by tradition, since their eggs and larvae are microscopic. Viruses are not cells at all, so they are acellular.

The second cut splits the cellular microbes again. A prokaryote has no nucleus. Its DNA sits loose in the cytoplasm. Bacteria and archaea are prokaryotes. A eukaryote keeps its DNA inside a nucleus, which is a pouch made of membrane. Fungi, protozoa and your own cells are eukaryotes.

Hold that line firmly. It pays off every time a drug is chosen. An antibiotic aims at a part only a bacterial cell has. The peptidoglycan wall is one such part. The bacterial ribosome is another. Hitting a target the microbe has and the host lacks is called selective toxicity. It is the whole reason drug therapy for infection works.

You are not sterile, and you were never meant to be. The microbes living on and in you are the microbiota. Those microbes plus all of their genes are the microbiome. Most of them help. Gut bacteria make vitamin K and some B vitamins. They crowd out invaders by filling the space and eating the food first, which is called competitive exclusion. They also help train a young immune system.

Flora come in two kinds. Resident flora stay for the long run. Transient flora are only passing through, picked up from a doorknob or a bedrail. Removing transient flora is the entire point of hand hygiene.

An opportunistic pathogen is normal flora that turns harmful. The microbe has not changed. Its chance has. It causes disease once it moves somewhere new, once rival flora are wiped out, or once defenses drop. Escherichia coli is harmless in the colon but causes a urinary tract infection in the bladder. Candida overgrows into thrush after antibiotics clear its bacterial rivals. Clostridioides difficile blooms into a severe colitis after antibiotics flatten the gut community.

Three words get used loosely in speech. In the clinic they mean three different things. Colonization means the microbe is present and multiplying on a surface, with no harm and no symptoms. Infection means it has invaded tissue and the body has begun to answer. Disease is one step further, with clear signs and symptoms and lost function.

That split is intensely practical. Picture a wound swab that grows bacteria. With no redness, no warmth and no fever, that is usually colonization. Antibiotics there do harm with no benefit. A pathogen is any microbe able to cause disease. Pathogenicity is that ability. Virulence is the degree of harm it can do.

A healthcare-associated infection, or HAI, is one picked up while receiving care. The common four are catheter-associated urinary tract infections, central-line bloodstream infections, surgical-site infections and ventilator-associated pneumonia. On any given day about one in thirty-one hospital patients has at least one. Most of that spread travels on hands.

Now the history. For most of history people blamed disease on miasma, which just means bad air. Others believed in spontaneous generation, the idea that life springs out of non-living matter. The germ theory of disease says something different. Specific microbes cause specific diseases. That idea is only about 150 years old.

Three people carry that story. Ignaz Semmelweis counted deaths in Vienna in the 1840s. Mothers died of childbed fever far more often on the ward staffed by doctors coming from autopsies. He ordered handwashing in a chlorine solution, and the death rate dropped sharply. He could not explain why, since germ theory did not exist yet. He is remembered as the father of hand hygiene. Louis Pasteur ended spontaneous generation with swan-neck flasks. He gave us pasteurization, a gentle heating that kills spoilage microbes. Robert Koch supplied the rigor with an orderly way to prove one microbe causes one disease.

Finally, names. Every living thing sits in one of three domains. Carl Woese worked the scheme out by comparing ribosomal RNA. Bacteria and Archaea hold the prokaryotes. Eukarya holds everything with a nucleus. Viruses are not cells, so they get no spot on this tree at all.

Every organism also gets a two-part Latin name. The system is binomial nomenclature, from Carl Linnaeus. The genus comes first and is capitalized. The species comes second and stays lowercase. The whole name is italic. After the first use, shorten the genus to one letter. So Staphylococcus aureus becomes S. aureus.

The groups worth memorizing

The three clinical states

The three domains

Sequences, in order

Koch's postulates are a four-step proof. The order is the point, so learn it as a chain.

1. The microbe is found in every case of the disease, and absent in healthy hosts.
2. It is isolated from a sick host and grown in pure culture.
3. The cultured microbe causes the same disease in a healthy host.
4. The same microbe is re-isolated from that newly diseased host.

The clinical ladder runs in one direction too. Each rung adds one thing.

1. Colonization · the microbe is present and multiplying. Nothing more.
2. Infection · add tissue invasion and a host response.
3. Disease · add signs, symptoms and lost function.

How to reason through the question types

Chapter 2Microscopy, Staining and the Microbiology Lab

What to know

Microbiology runs on the metric system. Start with the meter and divide by a thousand at each step. A millimeter is one thousandth of a meter. A micrometer is one thousandth of a millimeter. A nanometer is one thousandth of a micrometer. So each rung down is 1000 times smaller.

A red blood cell makes a handy yardstick at about 7 µm across. Most bacteria are smaller. A typical rod is about 0.5 µm wide and 2 to 3 µm long. A round coccus is about 1 µm. Yeasts and protozoa are eukaryotes, so they run from a few to tens of micrometers. Viruses are measured in nanometers, and most fall between 20 and 300 nm.

Your eye can tell two points apart only if they sit about 0.1 mm apart. That is 100 µm. A bacterium is about 1 µm wide, which is roughly a hundred times below the limit. Better eyesight will not help, and neither will brighter light. That hard limit has a name: resolution.

Every clinical lab leans on the compound light microscope. It is compound because it uses two lens systems in a row. Light starts at the illuminator in the base and travels up. The condenser gathers that light onto the specimen. The iris diaphragm sets how much light gets through, which controls contrast. The specimen rests on the stage. The objective lenses sit above it on a turning nosepiece and magnify first. The ocular magnifies second.

Total magnification is the ocular times the objective. Oculars are almost always 10×. Objectives come in a standard set: scanning at 4×, low power at 10×, high-dry at 40×, and oil immersion at 100×. So a 10× ocular with the oil lens gives 1000×.

Beginners chase magnification. Resolution is what decides whether an image is useful. Resolution is the smallest gap between two points that still lets you see them as separate. Blow up a blurry image and you only get a bigger blur, which is called empty magnification. The wavelength of visible light caps light-microscope resolution near 0.2 µm. That is fine for bacteria and useless for viruses, so viruses need an electron microscope.

To get the best out of the 100× lens, add a drop of immersion oil between the slide and the lens. Oil bends light the same way glass does, because its refractive index matches. Fewer rays scatter away, more reach the lens, and the image sharpens.

Living cells are mostly water and almost clear. Under plain light they nearly vanish. Staining fixes that by coating the cell in dye. First make a smear: spread a thin film on a slide and let it air-dry. Then heat-fix it. Fixing kills most organisms, glues them to the glass, and holds their shape. But heat-fixing is not sterilization. Mycobacteria and bacterial endospores can live through it.

Most bacterial dyes are basic dyes, which carry a positive charge. Methylene blue, crystal violet and safranin are all basic. The bacterial surface carries a negative charge, so it pulls them in.

A simple stain uses one dye. It shows size, shape and arrangement, and nothing more. A differential stain uses more than one reagent and sorts bacteria into groups. The Gram stain is the classic one. The acid-fast stain picks out the waxy-walled organisms, such as Mycobacterium tuberculosis, that a Gram stain misses. Special stains go after one structure, such as a capsule, an endospore or a flagellum.

Many special stains use negative staining. An acidic dye such as nigrosin, or a fine suspension like India ink, colors the background instead of the cell. The cell stays clear and glows against a dark field. Negative staining skips heat-fixing, so it does not warp delicate cells or shrink a capsule.

Now the stain that runs the show. Hans Christian Gram devised it in 1884. It works because of a real difference in the cell wall. Gram-positive bacteria have a thick outer layer of peptidoglycan, and that thick mesh acts like a sponge. Gram-negative bacteria have only a thin peptidoglycan layer, wrapped in an extra outer membrane.

Four reagents play off that difference, and the order never changes. Crystal violet is the primary stain, and it floods every cell purple. Gram's iodine is the mordant, and it binds the dye into a complex too big to slip out of a thick wall. Alcohol, or an alcohol-acetone mix, is the decolorizer. This is the step that truly splits the two groups. Alcohol tightens the thick wall, so the dye stays locked in. Alcohol also dissolves the Gram-negative outer membrane, and the dye washes straight out. Safranin is the pink counterstain, and it re-colors the now-colorless cells. So Gram-positive ends purple and Gram-negative ends pink.

A Gram stain can be read in under an hour, long before a culture matures. So it gives the first real clue for choosing empiric antibiotics. The report pairs the Gram reaction with shape and arrangement, and each pairing narrows the field.

A lab result is only as good as the specimen behind it. Sample the right site, meaning the infection and not the skin around it. Collect before antibiotics start whenever that is possible. Use the correct container and transport medium. Label it accurately at the bedside.

Back in the lab the sample goes onto a culture medium, usually a nutrient agar plate or a broth. Selective media hold back unwanted organisms. Differential media, such as blood agar or MacConkey, make different species look different.

To isolate one organism the lab uses a streak plate. The sample is dragged thinner and thinner until single cells sit far apart. Leave one overnight in a warm incubator and it divides into a visible mound of millions of identical clones. That mound is a colony, and it is a pure sample to work from. Its morphology is a first clue: size, shape, color, texture, and whether it breaks down blood in blood agar, which is hemolysis. Biochemical panels, automated analyzers or MALDI-TOF mass spectrometry then pin down the species.

The name is only half the answer. Someone still needs to know which drugs will kill it. The classic method is Kirby-Bauer disk diffusion. Small paper disks, each soaked with one antibiotic, sit on a lawn of the organism. Where a drug works it clears a zone of inhibition. Each zone is measured in millimeters and matched against standard breakpoints. You do not size it up against the disk next door. Other methods measure the minimum inhibitory concentration, or MIC, which is the lowest drug level that stops growth. Broth microdilution and a gradient Etest strip both report it. Results come back as S, I or R: susceptible, intermediate or resistant. That report is what lets a team de-escalate from a broad drug to one targeted drug.

The size scale, drawn

The tests worth memorizing

The Gram stain, in order

1. Crystal violet, the primary stain. Every cell turns purple.
2. Gram's iodine, the mordant. It traps the dye in a complex too big to escape a thick wall. Both groups are still purple.
3. Alcohol or alcohol-acetone, the decolorizer. Gram-positive cells stay purple. Gram-negative cells go colorless. This is the make-or-break step.
4. Safranin, the counterstain. The colorless cells turn pink. The purple cells do not change.

How to reason through the question types

Chapter 3Prokaryotic and Eukaryotic Cells

What to know

One line runs deeper than all the rest. Does the cell keep its DNA inside a membrane-bound nucleus? Cells that do not are prokaryotes. Cells that do are eukaryotes. That one answer comes with a whole package of other traits.

Prokaryotes are the bacteria and the archaea. They are small, with most running 0.2 to 2 µm across. They are plain inside, with one circular chromosome floating loose. Archaea are prokaryotes too, though their walls are made of other material, not peptidoglycan. Eukaryotes cover the fungi, protozoa, plants, animals and every human cell. They are usually ten to a hundred times wider, and far more involved inside.

The headline is what prokaryotes are missing. No nucleus. No other membrane-bound organelles either. No mitochondria, no endoplasmic reticulum, no Golgi apparatus. All of that chemistry happens out in the open cytoplasm, or against the inner face of the plasma membrane.

One more difference is easy to miss and huge for medicine. Both kinds of cell build proteins on a ribosome. Prokaryotes use a 70S ribosome. Eukaryotes use a larger 80S ribosome. That size gap is a gift, because a drug can be shaped to jam the 70S machine and ignore the 80S one.

Several layers wrap a bacterium. The plasma membrane, the cell wall, and in Gram-negatives an outer membrane. Together they are the cell envelope. Everything inside is cell contents: cytoplasm, ribosomes, nucleoid and plasmids.

The wall gives a bacterium its shape, but the plasma membrane is the layer that keeps the cell alive. It is a phospholipid bilayer, which means two sheets of fatty molecules. Water-loving heads face out and water-fearing tails tuck inside. It is selectively permeable, so it decides what crosses. Prokaryotes have no mitochondria, so the membrane takes one more job. It runs the electron transport chain and makes the cell's energy right there.

That makes the membrane a tempting drug target and a risky one. Bacterial and human membranes use similar chemistry. The polymyxins, such as colistin, punch holes in a membrane. They are held in reserve as last-resort agents against highly resistant Gram-negative bacteria, because they can also injure the kidneys.

Fill the membrane and you have the cytoplasm, a crowded water-based gel. Tens of thousands of 70S ribosomes hang in it, and whole families of antibiotics aim at them: aminoglycosides, tetracyclines and macrolides. Many bacteria also stock spare food in inclusions, which are small granules of stored carbon, phosphate or sulfur.

A prokaryote has not lost its DNA. The DNA is simply not gift-wrapped. The single circular chromosome coils into a dense zone called the nucleoid, with no membrane around it. So the DNA touches the cytoplasm directly, and transcription and translation can run in the same space at once.

Many bacteria also carry extra loops of DNA called plasmids. They are far smaller than the chromosome and sit apart from it. A cell can usually live without them. But plasmids often carry the genes that matter most in an outbreak: antibiotic-resistance genes, toxin genes, and the tools to pass those genes on. A plasmid can hop from one bacterium to another.

Outside the membrane, most bacteria wear a stiff wall made of peptidoglycan, also called murein. The human body holds none of it anywhere. Picture a chain-link fence wrapped around the cell. Long sugar chains run side by side, and short chains of amino acids tie them together. That cross-linking turns a floppy bag into a rigid mesh, strong enough to resist the cell's own osmotic pressure. The wall also sets the shape: rod, sphere or spiral.

This is exactly where the beta-lactam antibiotics work. Penicillins, cephalosporins and carbapenems block the enzymes that stitch the cross-links. The cell keeps growing, the wall can no longer hold, and the cell ruptures. Human cells have no wall to protect, so these drugs are among the safest we have.

Not all walls are built the same way. Gram-positive bacteria have a thick peptidoglycan layer on the outside, woven through with teichoic acids. It traps crystal violet, so the cell stays purple. Examples are Staphylococcus, Streptococcus, Enterococcus and Clostridioides.

Gram-negative bacteria have only a thin peptidoglycan layer, with a second outer membrane on top. Alcohol strips that outer membrane away and the purple dye washes out. The cell then takes the pink counterstain. Examples are Escherichia coli, Klebsiella, Pseudomonas and Neisseria.

A few organisms fit neither camp. Mycobacterium has a waxy wall that needs an acid-fast stain. Mycoplasma has no cell wall at all, so beta-lactams cannot touch it.

The Gram result predicts behavior, not just color. The extra outer membrane makes Gram-negative bacteria harder to kill, because it blocks many drugs from reaching the wall. It also carries lipopolysaccharide, or LPS, better known as endotoxin. When Gram-negative cells die and break apart, LPS spills into the blood. The immune system answers violently: fever, then blood pressure that drops away, and at the far end septic shock.

Now the outside of the cell. Some bacteria wrap themselves in a thick sticky layer called a capsule. A looser version is a slime layer. A cell does not need one to live, but where one exists it counts for a great deal. A capsule is a major virulence factor, because it is so slippery that immune cells struggle to grab and swallow the bacterium. That is resisting phagocytosis. The most dangerous strains of Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis are the ones with capsules. The capsule is also what several key vaccines aim at.

Flagella are long whip-like tails. They rotate like a propeller and push the cell through liquid. Bacteria do not wander at random. They steer toward nutrients and away from toxins, and that directed movement is chemotaxis. Motility helps organisms like E. coli and Pseudomonas climb a urinary catheter or reach fresh tissue.

Two shorter appendages finish the outside, and the naming is easy to mix up. Fimbriae are short hair-like fibers, and there are many of them. They attach the cell to surfaces and to host cells. Attachment is the first step of nearly every infection, since a microbe that cannot stick gets flushed away. Fimbriae are how E. coli grips the bladder lining, and they anchor biofilms on catheters and implants.

Pili are usually longer and far fewer, often one or two per cell. The sex pilus, also called the conjugation pilus, builds a bridge to another bacterium and pulls it close. The two cells then swap DNA in conjugation. This is one of the main ways resistance plasmids move.

Last come the spores. A handful of Gram-positive bacteria can do something no other microbe can. The main groups are Bacillus and Clostridioides or Clostridium. When conditions turn hostile they seal a copy of their DNA inside an armored capsule called an endospore. The spore is not a way to reproduce. One cell makes exactly one spore. It is a way to wait out disaster, and it can persist for years.

The normal growing bacterium is the vegetative cell. Under stress it begins sporulation. It copies its chromosome, walls off a patch of cytoplasm around that DNA, then wraps the patch in layer after layer. A dense core stripped of most of its water. A thick cortex. A tough protein coat. The rest of the cell then breaks down and releases the spore. When nutrients return the spore germinates, the coat cracks, water rushes in, and it grows back into a vegetative cell.

That last part matters most. The vegetative cell causes active disease, and ordinary antibiotics and disinfectants can kill it. The spore is the form that survives to start the cycle over.

A spore holds very little water and its armor comes in layers. Together those two facts make it extraordinarily hard to kill. It shrugs off heat, drying, radiation and most chemical disinfectants, including alcohol-based hand rubs. So for a case of C. difficile the rule is soap-and-water handwashing, which physically washes spores away, plus a sporicidal cleaner such as diluted bleach for surfaces. And because spores survive boiling, true sterilization of instruments needs an autoclave, which is pressurized steam at 121°C.

The two cell envelopes, drawn

The structures worth memorizing

The drug classes worth memorizing

Sequences, in order

Sporulation and germination make a loop. Learn the loop, because every infection control rule about spores hangs on it.

1. A vegetative cell grows and divides normally. This is the form that causes disease and dies to drugs.
2. Stress arrives, such as starvation, and sporulation begins. The chromosome is copied.
3. A patch of cytoplasm is walled off around that DNA copy.
4. The patch is wrapped in layers: a dry dense core, a thick cortex, then a tough protein coat.
5. The rest of the original cell breaks down and releases the endospore. It is dormant and can wait for years.
6. Nutrients return and the spore germinates. The coat cracks, water rushes in, metabolism restarts.
7. It grows back into a vegetative cell, and the loop closes.

Working from the outside of a Gram-negative cell inward, the layers come in this order.

1. Outer membrane, studded with LPS and gated by porin channels.
2. Periplasmic space, the gap between the two membranes.
3. Thin peptidoglycan wall, sitting inside that gap.
4. Plasma membrane, which handles transport and energy.
5. Cytoplasm, holding the 70S ribosomes, the nucleoid and any plasmids.

How to reason through the question types

One pageCram sheet